Subjects

Notes

Abstract:

Vitamin B12 (B12) status of young adults has been considered adequate based on estimated intakes that met the RDA; however, few studies in the US have evaluated B12 status of young adults using a panel of B12 biomarkers. Vitamin B12 deficiency impairs neurological function and increases other health-related risks. Early detection and determination of whether B12 deficiency is due to dietary insufficiency, a genetic abnormality, or malabsorption are critical to effective treatment. The aims of the first study were to compare B12 status using numerous biomarkers in young adult non-supplement users consuming vegetarian and omnivorous diets, determine the level of intake associated with optimal B12 status, and determine if the transcobalamin (TC) 776C → G polymorphism affected B12 metabolism. Blood samples were collected for determination of holo-TC, B12, methylmalonic acid (MMA), and homocysteine (Hcy) (n = 388). Dietary B12 intake was assessed using a food frequency questionnaire. A surprisingly high incidence of B12 deficiency was observed in both vegetarians and omnivores. Relative to omnivores, vegetarians had a higher rate of B12 deficiency, with lower B12 and higher MMA concentrations. Vitamin B12 status improved with B12 intake above the RDA. No differences were detected between TC 776C → G genotypes for any biomarkers. In the second study the magnitude and patterns of post-absorption changes in several B12 biomarkers were assessed. Subjects (n = 21) had blood drawn at 17 intervals over three days with administration of three 9 microg doses of B12 at 6 hour intervals on day one. Mean B12, holo-TC, TC saturation, and the ratio of holo-TC to B12 increased significantly from baseline at hour 24 only. In conclusion, a high incidence of impaired B12 status was observed in otherwise healthy young adults. The data suggest that further assessment of the adequacy of the B12 RDA is warranted.
Measurement of multiple B12 biomarkers may provide a more accurate assessment of B12 status than measurement of one biomarker alone. Holo-transcobalamin appears to be a sensitive indicator of B12 absorption and a holo-TC based absorption test should involve measurement at 0 and 24 hours. No effect of the TC 776C → G polymorphism was detected.

Subjects

Notes

Abstract:

Vitamin B12 (B12) status of young adults has been considered adequate based on estimated intakes that met the RDA; however, few studies in the US have evaluated B12 status of young adults using a panel of B12 biomarkers. Vitamin B12 deficiency impairs neurological function and increases other health-related risks. Early detection and determination of whether B12 deficiency is due to dietary insufficiency, a genetic abnormality, or malabsorption are critical to effective treatment. The aims of the first study were to compare B12 status using numerous biomarkers in young adult non-supplement users consuming vegetarian and omnivorous diets, determine the level of intake associated with optimal B12 status, and determine if the transcobalamin (TC) 776C → G polymorphism affected B12 metabolism. Blood samples were collected for determination of holo-TC, B12, methylmalonic acid (MMA), and homocysteine (Hcy) (n = 388). Dietary B12 intake was assessed using a food frequency questionnaire. A surprisingly high incidence of B12 deficiency was observed in both vegetarians and omnivores. Relative to omnivores, vegetarians had a higher rate of B12 deficiency, with lower B12 and higher MMA concentrations. Vitamin B12 status improved with B12 intake above the RDA. No differences were detected between TC 776C → G genotypes for any biomarkers. In the second study the magnitude and patterns of post-absorption changes in several B12 biomarkers were assessed. Subjects (n = 21) had blood drawn at 17 intervals over three days with administration of three 9 microg doses of B12 at 6 hour intervals on day one. Mean B12, holo-TC, TC saturation, and the ratio of holo-TC to B12 increased significantly from baseline at hour 24 only. In conclusion, a high incidence of impaired B12 status was observed in otherwise healthy young adults. The data suggest that further assessment of the adequacy of the B12 RDA is warranted.
Measurement of multiple B12 biomarkers may provide a more accurate assessment of B12 status than measurement of one biomarker alone. Holo-transcobalamin appears to be a sensitive indicator of B12 absorption and a holo-TC based absorption test should involve measurement at 0 and 24 hours. No effect of the TC 776C → G polymorphism was detected.

Holo-transcobalamin concentration can be used to assess Bl2 absorption.

Specific aim: To determine if holo-TC concentration increases measurably in response to

Bl2 supplementation within a 24 hour time period.

Beta-IgandR

Corn n Ring H2N I HCTs" c

NH OH

Alpha-ligand

Figure 1-1 Structure of vitamin Bl2. Modified from Stabler (35) p. 22

Ileum

~1% diffusio

Enterocyte

Figure 1-2 Overview of vitamin Bl2 (Bl12) absorption. (1) Food bound Bl2 is released in the
acidic environment of the stomach. (2) Free Bl2 binds to haptocorrin and the
complex travels to the duodenum. (3) Pancreatic proteases degrade HC. (4) Free
Bl2 binds to intrinsic factor, which is synthesized in the gastric parietal cells. (5) The
Bl2 IF complex to travel to the ileum and is transferred across the ileal epithelium
via receptor mediated endocytosis, along with 1% passive diffusion. (6) In the
enterocyte, intrinsic factor is degraded by the lysosome. (7) Transcobalamin binds
Bl2 at some point after release from intrinsic factor, this may occur in the enterocyte.

supplementation. Data from the current investigations support the use of holo-TC as an indicator

of Bl2 absorption though further research is needed before a clinically reliable test could be

developed.

APPENDIX A
SUBJECT PHONE SCREENING FORM

Introduction

I am calling in regard to your interest in our nutrition study; do you have a few minutes right
now?

This is a UF Nutrition department study and involves coming in one morning for about I hour
for a fasting blood sample, we take about 1 V/2 Ounces of blood, and you only need to fast 8 hours.
We will give you a breakfast snack right afterward, and then give a brief explanation of a food
frequency questionnaire you will be taking home. You will be asked to mail it back in the
provided envelope, and once we receive the questionnaire you would get paid the $50. I just
have to ask you some questions to see if you are eligible for our study and to get background
information, OK?
How old are you? M~ust be 18-49
Do you smoke? M~ust answer no
Are you pregnant or breastfeeding? M~ust answer no
Do you take any prescription medications other than oral contraceptives? M~ust answer no

If not ~I ithrin the age range or if they answer yes to any above questions end call 11 irl.
I am very sorry, but you do not meet our exclusion criteria, but thank you for your interest.

Now I just have a few questions about your diet to see what specific category of our study
you would fit in to. Please answer as best you can, estimates are ok and consider all
instances of when you might eat the items I will ask about, even if only occasionally.

Do you take a multi-vitamin, complex, red star nutritional yeast, or any other supplement
or additive ever?

If they takettttt~~~~~~~tttttt a multivitamin,~~ttt~~~ttt~~~ B complex, red star nutritional yeast, complete the session through
all diet info only. Conclude by confirming thelir name and saysss~~~~~ssssing "This has been a preliminary
screening call, your information will be reviewed by the principal investigator based on need,
and our selection criteria at this time. If you are chosen you will be called again to schedule an
appointment over the next two weeks. Thank you very much for your interest and your time.

Do you eat breakfast cereals? (Ifso) What Kind do you eat mostly?

If they eat a 100% fortified cereal or eats a 50% cereal daily complete the through all diet info
but do not record. Conclude by confirming their name and saying "This has been a preliminary
screening call, your information will be reviewed by the principal investigator based on need,
and our selection criteria at this time. If you are chosen you will be called again to schedule an
appointment over the next two weeks. Thank you very much for your interest and your time.

If the interviewee fulfills all selection criteria continue with the questionnaire, record info on
moderate/non-fortified cereal consumption

Do you eat
o Yes
o No

breakfast cereals?

Name/Brand

Quantity

Frequency

Are you a vegan, vegetarian or meat eater?
Vegan this means you eat NO animal derived foods intentionally (if they eat small amt like in
cake then OK)
Vegetarian this means you eat NO beef, chicken, turkey, pork, or fish
How often do you eat ...

o No
0 Yes; How long ago did you make changes and what changes did you make?

NO YES

Do you consume alcoholic beverages?
> How often/quantity
Health Information

I am going to ask you a few questions about your health to determine if you are eligible for
our study. I will be recording this information, but it will be kept confidential and is this
ok with you?
Height: Weight:

If you are selected to participate in this study are you willing to sign an informed consent
understanding we have access to medical information on you?
o Yes
o No

Demographic Information

What is your birth date?

Month Day Year

How would you describe your race or ethnic background?
o White
0 Black or African American
0 American Indian or Alaska Native
0 Hispanic or Latino
0 Asian
0 Native Hawaiian or Other Pacific Islander
o Other

What is the highest level of school or training that you have completed? [Circle only one
response]

Grade school
High school
Technical school or college
Graduate or professional
Don't know X

1 VITAMIN B12 STATUS AND ABSORPTION USING HOLO-TRANSCOBALAMIN IN YOUNG MEN AND WOMEN By KRISTINA VON CASTEL-ROBERTS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006

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2 Copyright 2006 by Kristina von Castel-Roberts

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3 To my father Gerard David von Castel-Dunw oody and my uncle Gnter von Castel; they left this world too early but will live in my heart forever.

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4 ACKNOWLEDGMENTS I would like to thank my committee members, doctors Lynn B. Bailey, Gail P.A. Kauwell, Jesse F. Gregory III, and Lee McDowell for their guidance and support. I would particularly like to thank Dr. Bailey and Dr. Kauwell for their dail y encouragement during th is exciting endeavor. Their dedication and achievement in the field of science has given me high standards to follow and has driven me to push myself to the best of my potential and beyond. I would like to thank Dr. Gregory for the contribution of his scientif ic and technical knowledge and Dr. McDowell for making me aware of how my animal nutrition education can help me better understand human nutrition. I would like to express my gratitude to the members of our laboratory team, especially David Maneval, Amanda Brown, Claire Edgem on, and Dr. Karla Shelnutt. It was their combined effort that made it possible to succ essfully conduct two human studies, teaching me the value of teamwork. I owe tremendous thanks to my friends a nd family who supported me through the tough spots, whether a few miles or a few hundred miles away. Their help and support allowed me to keep my farm standing and my horses healt hy, plan my wedding, get married, escape on my honeymoon, and still have fun while pursuing my degree. I would partic ularly like to thank those who have been with me from the start of my Gator career, Karen Knight, Christina Stortz and Tiffany Tooley, and my friend turned-siste r Revati Roberts. Spec ial thanks go to my mother and father Alice von Castel Dunwoody and Gerard von Castel Dunwoody, for their unending and unconditional love an d support, and for believing I c ould do anything I truly set my mind to. Finally, my deepest love and a ppreciation go to my husba nd and best friend Nando David Roberts for the continuous support, love, and devotion he has given me.

12 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy VITAMIN B12 STATUS AND ABSORPTION USING HOLO-TRANSCOBALAMIN IN ADULT MEN AND WOMEN By Kristina von Castel-Roberts December 2006 Chair: Lynn B. Bailey Major Department: Food Science and Human Nutrition Vitamin B12 (B12) status of young adults has been considered adequate based on estimated intakes that met the RDA; however, few studies in the US have evaluated B12 status of young adults using a panel of B12 biomarkers. Vitamin B12 deficiency impairs neurological function and increases other health -related risks. Early detecti on and determination of whether B12 deficiency is due to diet ary insufficiency, a genetic abno rmality, or malabsorption are critical to effective treatment. The aims of the first study were to compar e B12 status using numerous biomarkers in young adult non-supplement users consuming vegeta rian and omnivorous diets, determine the level of intake associated with optimal B12 status, and determine if the transcobalamin (TC) 776C G polymorphism affected B12 metabolis m. Blood samples were collected for determination of holo-TC, B12, methylmalonic aci d (MMA), and homocysteine (Hcy) (n = 388). Dietary B12 intake was assessed using a food fr equency questionnaire. A surprisingly high incidence of B12 deficiency was observed in bo th vegetarians and omnivores. Relative to omnivores, vegetarians had a higher rate of B 12 deficiency, with lower B12 and higher MMA concentrations. Vitamin B12 stat us improved with B12 intake a bove the RDA. No differences were detected between TC 776C G genotypes for any biomarkers.

PAGE 13

13 In the second study the magnitude and pattern s of post-absorption ch anges in several B12 biomarkers were assessed. Subjects (n = 21) had blood drawn at 17 intervals over three days with administration of three 9 g doses of B12 at 6 hour inte rvals on day one. Mean B12, holoTC, TC saturation, and the ratio of holo-TC to B12 increased significantly from baseline at hour 24 only. In conclusion, a high incidence of impaired B 12 status was observed in otherwise healthy young adults. The data suggest th at further assessment of the adequacy of the B12 RDA is warranted. Measurement of multiple B12 biomarkers may provide a more accurate assessment of B12 status than measurement of one biomarker alone. Holo-transcobalamin appears to be a sensitive indicator of B12 absorption and a holo-TC based absorption test should involve measurement at 0 and 24 hours. No effect of the TC 776C G polymorphism was detected.

PAGE 14

14 CHAPTER 1 INTRODUCTION Vitamin B12 History Vitamin B12 (B12) is one of the thirteen esse ntial vitamins that humans must obtain from their diet. Vitamin B12 was the last vitamin to be discovered, due in part to the lack of a suitable animal model in which to study the B12-related disease pernici ous anemia (1). Pernicious anemia, which literally means fatal anemia, has been reported in medical records as far back as the early 1800s, although the condi tion was likely responsible for deaths well before then. The earliest studies of patients with pernicious anemia led to the know ledge that the disease was due to some ailment of the stomach; although no treatm ent was available and most patients died from the disease (2). In the early 20th century, Minot and Murphy determ ined that feeding liver to patients with pernicious anemia improved thei r condition, a discovery for which they received the Nobel prize in 1934 (3). Castle conducted a series of experiments comparing the treatment of pernicious anemia patients with partially dige sted beef, or beef inc ubated in gastric juice, versus undigested beef. Patients receiving the pre-digested beef improved while those receiving undigested beef did not, suggesting that some (intrinsic) factor w ithin gastric juice interacted with the unidentified (extrinsic) factor in the beef (4-7). The final identification of this extrinsic factor was delayed until 1945 when it was discovered that the anti-anemia substance was required by Lactobacillus locus finally providing a useful labo ratory model (8). Vitamin B12 was crystallized in 1948 and was qui ckly identified as th e illusive extrinsic anti-anemia factor (9-12). After these discoveries, B12 research proceeded rapidly as did our understanding of the vitamins functions.

PAGE 15

15 Chemistry Vitamin B12 is a complex water-soluble mo lecule with a molecular weight of 1655.38 daltons. The molecule is comprised of a c obalt atom centered in a corrin ring, with two coordinating ligands. The 5,6-dimethylbenz imidazole component is linked to the -axial position of the cobalt, and a vari able ligand is linked to the -axial position (F igure 1-1) (13). Known ligands include CH3 (methylcobalamin), 5-deoxyade nosyl (adenosylcobalamin), OH (hydroxylcobalamin), and CN (cyanocobalamin). Cyanocobalamin, the synthetic form of B12 included in vitamin supplements and fortified foods, is converted to methylcobalamin or adenosylcobalamin, the two coenzyme forms of the vitamin. Methylcobalamin is the primary form found in human plasma making up 60 to 80% of total cobalamins (14). Nomenclature for B12 Binding Proteins The nomenclature for B12 binding proteins in the gastrointestinal tract and plasma has changed over time, and both the new and old terms are used in current literature. The term Rprotein was originally used to differentiate B12 binding proteins, which move rapidly upon electrophoresis, from intrinsic fact or (IF), which moves slowly. In testinal R-protein, now termed haptocorrin (HC) because of its ability to bind corrins other than B12, is also found in saliva, bile, and plasma. Transcobalamin (TC) I, II, and III were terms used to identify the plasma B12 binding proteins; however, further investigation pr oved that TC I and III we re isoforms of HC, which differed only by carbohydrate content. The te rm transcobalamin II was used to identify the B12 binder that participated in delivery of B 12 to cells, but now it is s imply referred to as TC (15).

PAGE 16

16 Absorption The absorption of B12 is primarily an active receptor mediated process that uses several different transporters (Figure 1-2). Vitamin B12 is bound to proteins in foods and must be liberated by the action of pepsin and hydrochlor ic acid (HCL) in the stomach in order for absorption to occur. Reduced gastric pH, as ofte n seen in adults over the age of 50 y and with chronic antacid use, impairs brea kdown of the protein matrix and ultimately results in reduced B12 absorption (16, 17). Once B12 is released fro m the protein matrix, it binds to HC enabling it to travel to the duodenum where pancreatic pr oteases degrade HC. In the duodenum, free B12 binds to IF, a glycoprotein synt hesized and secreted from gast ric parietal cells. The IF-B12 complex is resistant to attack from pepsin, c hymotrypsin, and intestinal bacteria, allowing the complex to travel to the ileum intact, where it is transferred across the ileal epithelium viareceptor mediated endocytosis. This ileal recep tor (cubilin) only recognizes the IF-B12 complex, so that free B12 can not cross the membrane in this manner (18, 19). Although passive diffusion of B12 across the epithelium does occur at a rate of 1% of any B12 dose, B12 is primarily absorbed by active transport (14). Once in the enterocyte, IF is degraded by the lysosome. Transcobalamin plays an essential role in B 12 absorption, binding B12 at some point after release from IF and appearance in the blood as holo-TC. The exact mechanism by which B12 binds to TC is under debate, however, it is hypot hesized that binding occurs in the enterocyte (20-22). Holo-transcobalamin can be detected in the blood 3 hours af ter B12 intake, with maximum absorption occurring 8 to 12 hours after intake. Cellular up take occurs within minutes (23, 24). Transport and Cellular Uptake Transport of B12 in circulatory system and into the cells of target tissues is dependent on two binding proteins, TC and HC. Each protein has only one bindi ng site with a high affinity

PAGE 17

17 (Kd = 10-10 to 10-17) for the various chemical forms of B12 (25). Transcobalamin is a 43 kDa non-glycosylated protein found in plasma and in various cells in cluding endothelial cells (22). Numerous tissues contain TC mR NA, including kidney, heart, live r, and leukocytes; however the specific cell type in which TC is synthesized is unknown (26). Transcobalamin is required for transport of B12 into the cell since only the B12 bound to TC is taken up by cell surface receptors. For this reason, the holo-TC fraction of serum B12 is the only component that is considered biologically active (27-29). Th e TC receptor (TC-R) is a 50 kDa heavily glycosylated protein, that binds both holo-TC and apo-TC (TC with no B12 bound) (26). In the cytoplasm, lysosomes break down the holo-TC co mplex making free B12 available for metabolic processes. Holo-transcobalamin constitutes on ly 20% of plasma B12, the remaining 80% is bound to HC (13, 30). Haptocorrin is a heavily-g lycosylated, 70 kDa protein found in various biological fluids including saliva, bile, and bloo d. Although the majority of plasma B12 is bound to HC (holo-HC), holo-HC cannot be used by the cells as there are no rece ptors for this complex (15). The function of HC is unclear and is still debated (15, 31, 32). Storage and Turnover The main storage tissues for B12 are the li ver and muscle, which contain approximately 60% and 30%, respectively, of the bodys total B12. High concentrations also are found in the pituitary, kidney, heart, spleen and brain. Inte restingly, although B12 is enzyme bound in most tissues, the kidney maintains a pool of free B12, which can be used to maintain plasma B12. When B12 intake is high, uptake of B12 in to the kidney increases; when plasma B12 concentrations are low, B12 is released first from the kidney (33, 34). Mean total body stores range from 2 to 5 mg with a half life of 340 to 400 days (14). Vitamin B12 is excreted only in the free form in the urine and bile at a rate of 0.1 to 0.2% (2 to 5 g) of total body reserves per day (35). Enterohepatic recirculatio n is very efficient and

PAGE 18

18 helps reduce total loss of B12 (13). Up to 75% of biliary B12 is actively re absorbed in the ileum, so that very little is excreted in the feces, effectively conservi ng this essential nutrient (14). Additionally, because the kidney is rich in TC -R, B12 stored in the kidney is efficiently reabsorbed back into circul ation reducing urinary losses. Biochemical Reactions In humans and other higher animals, B12 serves as a coenzyme for two metabolic processes, the conversion of me thylmalonyl-CoA to succinyl-CoA as adenosylcobalamin and the remethylation of homocysteine (Hcy) to methi onine as methylcobalamin (13, 35). In succinylCoA synthesis, adenosylcobalamin undergoe s homolytic cleavage by the action of Lmethylmalonyl CoA mutase forming cob(II)alami n and a 5'-deoxyadenosyl radical. Radical formation allows the rearrangement of the Lmethylmalonyl-CoA molecule to form succinylCoA. The Hcy remethylation process is an im portant component of overall one carbon metabolism. Methionine synthase (MS) catalyzes the remethylation of Hcy to methionine with the associated B12 acting as a methyl carrier. Methionine synthase c ontains separate binding domains for Hcy, 5-methyltetrahydrofolate (5-CH3THF), B12, and S-adenosylmethionine (SAM). Vitamin B12 in its reduced active st ate as cob(I)alamin is remethylated by 5-CH3THF and the methyl group can again be donated to Hcy (Figure 1-3). Methionine is of great biological importance because it is the precurs or of SAM the major methyl donor in over 100 biochemical reactions (36, 37). Daily Requirement The Dietary Reference Intakes (DRI) for esse ntial nutrients are guidelines for estimating the average vitamin and mineral intake needed to maintain health ( 38). The DRIs include Estimated Average Requirement (EAR), Ade quate Intake (AI), Recommended Dietary

PAGE 19

19 Allowance (RDA) and Tolerable Upper Intake Leve l (UL). The RDA for nut rients is calculated from the EAR and is defined as the average daily intake required to meet the needs of most individuals in the defined age br acket (38). The daily requirement of B12 is relatively low in comparison to other essential vitamins, with an EAR and RDA for adult men and women (19 to 50 y) of 2 g/d and 2.4 g/d, respectively. Estimates for the EAR for adults are based on the level of intake needed to maintain normal he matological status and a serum B12 concentration above 150 pmol/L minus the amount conserved in daily B12 turnover. Data were gathered primarily from patients with pernicious anem ia in remission who were receiving regular intramuscular (IM) injections of B12. Studies of patients with pernicious anemia reported that IM doses of 0.8 to 1.7 g/d were sufficient to ma intain normal hematological parameters. From these studies an average of 1.5 g/d was estim ated to be the B12 requirement. The final calculation of the EAR in hea lthy adults with normal B12 ab sorption was calculated as 1.5 g/day minus 0.5 g/d (the estimated amount of B12 reabsorbed) with a correction to account for an estimated bioavailability of 50% (38). The RDA was then calculated as the EAR (2.0 g/d) plus twice the coefficien t of variation (CV) for 97 to 98% of the population, or 120% of the EAR. Recommendations for children are based on B12 concentrations in milk for infants and are extrapolated down from adult requirement s for children up to age 18 y. Adults over the age of 50 y have the same RDA as younger adults ; however, due to an ag e-related reduction in gastric pH, it is recommended that older adults ob tain most of the RDA from fortified foods and B12 supplements (23). Dietary and Supplemental Sources Vitamin B12 is synthesized only by microorganism s in bacteria rich environments such as the intestinal tracts of animals. Some species ha ve sufficient microbial s ynthesis of B12 to meet

PAGE 20

20 their biological need without any additio nal dietary B12. Although humans have B12 synthesizing bacteria in their in testinal tract, they ar e primarily found in the colon where little absorption takes place making B12 an essential nut rient for humans. Natural dietary sources of B12 are limited to foods of animal origin. Th e liver and kidney stor e large amounts of B12, therefore, these organ meats are the richest dietary sources of B12 (24 to 122 g/100 g). Other more commonly consumed food sources are red meat (0.55 to 3.64 g/100 g), poultry (0.32 to 0.379 g/100 g), fish (1.9 to 21.2 g/100 g), eggs (0.09 to 9.26 g/100 g), and milk products (0.06 to 1.71 g/100 g) (14). Vitamin B12 al so can be obtained in the following: (a) supplements; (b) B12-fortified foods such as cereals and meal re placement bars and drinks; (c) B12fortified vegetarian products such as soy milk; and (d) B12fortified meat substitutes and frozen meal entrees made from wheat gluten or soybeans; and some fermented food products (39). In the US, foods of animal origin are a common part of the diet, and the estimated average daily intake of B12 from food sources is 3 to 5 g/d on average (38). The largest percentage of dietary B12 in the US diet comes from mixed foods (16 to 19%) which includes non-beef meats, poultry, and fish; a substantial por tion also comes from beef (12 to 15%), and milk products (11 to 15%). In the case of vegeta rians, who are estimated to repr esent up to 25% of US women of reproductive age, dairy and egg products for laco to-ovo-vegetarians and da iry products for lactovegetarians, are the sole sources of B12 if supplements or for tified foods are not consumed. Individuals who exclude some or all animal -derived foods and do not add B12 fortified foods to their diet are at increased risk fo r developing B12 deficiency (40-42). Non-meat animal-derived sources of B12 including dairy pr oducts and eggs can contribute significantly to B12 intake (38), but are excluded from the diets of strict vegeta rians (vegans). The extent to

PAGE 21

21 which animal-derived foods are excluded from the diets of self-described vegetarians may determine the effect on B12 status. For example, one serving (4 oz) of beef can provide the RDA for B12 (2.4 g/d), while one serving of chicken (4 oz) provides only 12% of the RDA. The B12 content of fish varies by species; 1 se rving (4 oz) of grouper provides 25% of the RDA, while tuna and herring provide as much as 500% of the RDA per serving (1 to 4 oz). Dairy products also provide variable amounts of B12 w ith 8 to 50% the RDA per serving (2 to 8 oz) (39). Vitamin B12 Status Assessment Vitamin B12 Concentration In clinical settings, serum B12 determin ation is the primary method for assessing B12 status (35). In the genera l US population, the mean serum B12 concentration for healthy individuals over four years of age is 381 pmol/L (43). Reliance on serum B12 as the sole diagnostic tool may lead to a false diagnosis since not all individuals with low values are deficient and a normal serum concentration may or may not indicate adequa te B12 status (44). This is due in part to the manner in which B 12 is metabolized and stor ed in the body. Because some tissues, such as the liver and kidney, can store a relatively large amount of B12, total body depletion takes years; however, some cells with lower storage capacity may become B12 deficient while circulating B12 is still in the low normal range. In these cells, B12 dependant enzyme function may become impaired causing elevated MMA and Hcy. Currently, clinical B12 deficiency is classified as serum c oncentrations < 148 pg/mL; however, a significant percentage of patients with cl inical symptoms of B12 defici ency who respond to B12 therapy have serum B12 concentrations in the low-nor mal (148 to 221 pg/ml) range. To enhance the diagnostic value of serum B12 concentrations, a dditional status indicators should be evaluated.

PAGE 22

22 Holo-transcobalamin Concentration Measurement of holo-TC is considered a functional indicator of B12 status because only the B12 bound to TC can be taken up by cell receptor s for use in intracellu lar metabolic reactions (27, 28, 45, 46). In contrast, serum B12 consists of holo-TC (~20%) and holo-HC (~80%), the latter can not be used by cells since it lack s known cellular receptors. Evidence supports the conclusion that holo-TC concentr ation responds more rapidly to changes in B12 intake than other indices of B12 status ( 28). Bor et al. (47) reported that oral B12 treatment (400 g/d) resulted in a highly significant maximal increase (+ 54%) in holo-TC after 3 days, in contrast to serum B12, which responded with a smaller initia l change (+28%) and a sl ower graded increase over time. Routine measurement of holo-TC as an index of B12 status is now possible since technical problems associated with the analytical procedure have been successfully addressed (Holo-TC RIA, Axis-Sheild) (45, 48). Loikas et al. (49) confirmed the su itability of the holo-TC RIA for use in a clinical laboratory, determin ed reference values for the method (37 to 171 pmol/L), and confirmed that low holo-TC concentrations (< 35 pmol/L) were associated with other biochemical indicators of low B12 status. Methylmalonic Acid Concentration When B12 status is low the conversion of methylmalonyl-CoA to succinyl CoA is impaired; as methylmalonyl-CoA accumulates, it is converted to methyl malonic acid. This alteration in metabolism results in a measur able increase in MMA. Methylmalonic acid concentration is a highly specific diagnostic i ndicator of B12 status because, unlike the MS reaction that requires both B12 and folate, no ot her nutrient is required for the methylmalonyl CoA mutase reaction (50-52). A nor mal serum MMA concentration is 271 nmol/L, with reported reference ranges for serum MMA concentr ation of ~50 to 400 nmol/L (53, 54). Serum MMA concentration also provides diagnostic info rmation when it is obtained before and after

PAGE 23

23 B12 supplementation in B12 deficient individua ls. Moelby et al. ( 52), like previous investigators, reported a marked decline in se rum MMA concentration to normal one month after treatment with B12 (55). Homocysteine Concentration Homocysteine concentration is inversely associated with B 12 status, and may or may not be elevated in individuals with low B12 status. Individuals that have el evated Hcy due to a B12 deficiency will respond to B12 supplementation (55, 56). Traditionally the cut-off for normal Hcy concentration has been 14 mol/L; however, due to the im plementation of mandatory folate fortification in the US, Hcy concentr ations within the popul ation have decreased significantly (57, 58). In a populat ion-based study, Selhub et al. ( 56) reported that plasma Hcy concentration was inversely associated with B12 status, and mean Hcy concentration was significantly higher in indi viduals in the lowest compared to the highest decile for plasma B12 concentration (15.4 and 10.9 mol/L, respectively). Mezzano et al. (59) evaluated plasma Hcy concentrations and response to B12 therapy in a group of vegetarians with low B12 status (baseline mean serum B12 concentration 110 pmo l/L) with elevated plasma Hcy concentration. Following intramuscular injection with B12, seru m B12 concentration incr eased to 392 pmoL/L and mean plasma Hcy concentration dropped significantly (12.4 to 7.9 mol/L). Unlike MMA, Hcy concentration is not a specific indicator of B12 status. Because the folate derivative, 5-CH3THF is the methyl donor in the conversion of Hcy to methionine, low folate status also can lead to an elevation in Hcy concentration.

PAGE 24

24 Vitamin B12 Deficiency Etiology Vitamin B12 deficiency may occur due to dietary restriction, malabsorption, or disturbances in transport or cellular uptake. Malabsorption of B12 can be caused by several physiological and congenital defects. Pernicious anemia is a dis ease of the autoimmune system in which antibodies to parietal cells and IF develop leading to a complete lack of IF and an inability to absorb B12. Individu als with this condition can be gi ven IM B12 injections to meet B12 requirements, bypassing the absorption process (35). The elderly popul ation is at a higher risk of B12 malabsorption due to the age-re lated decrease in stomach acid. The acidic environment in the stomach is required for th e release of B12 from food, and a significant decrease in hydrochloric acid can impair the process leadi ng to increased excretion and decreased absorption. In such cases the daily B12 requirement must be met with supplemental B12 (35). Clinical Abnormalities Depleted B12 status may take years to develop in individuals with im paired absorption or inadequate intake and an indivi dual may have marginal B12 stat us prior to developing severe clinical symptoms such as megaloblastic anemia and irreversible neurological damage (32). Development of B12 deficiency begins with depletion of serum B12, followed by cellular deficiency and biochemical changes including elevated Hcy and MMA concentrations (32). Neurological abnormalities affecting physical reflex es, stamina, and mental attributes including memory and behavioral changes may accompany a moderate B12 deficiency (60-62). In addition, the risk of inadequate B12 intake to a developing fetus, should pregnancy occur, is of great concern for women of repr oductive age. Infants born to mo thers with a B12 deficiency have been reported to suffer devastating symp toms including growth retardation, delayed

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25 psychomotor development, and in some instan ces, permanent effects on the brain (63-66). A B12 deficiency may increase the risk for birth defects as illustrated by the well documented independent role for B12 in the etiology of neur al tube defects (NTDs) (67-73). These studies have provided evidence that even small reducti ons in serum B12 concentrations within the normal range may be associated with a significan tly increased risk for NT Ds. Afman et al. (74) measured the plasma concentrations of B12, Hc y, and the apoand holoforms of TC in NTD case mothers and in control women. Low plasma holo-TC concentration wa s associated with a 3-fold increased risk for having a child with an NTD, while a low percentage of B12 bound to TC (TC saturation) was associated with a 5-fold increased risk. Vegetarianism Vegetarians are at increased risk for deve loping a B12 deficiency since B12 is only naturally present in animal-derived products (i.e., meat, eggs, dairy). Th ere is much variability in the amount of B12 consumed by individuals characterized as vegetarians. This classification includes those who consume diet s completely devoid of all animal-derived products (vegans), including meat, fish, dairy, an d eggs, as well as those who exclude meat but consume either dairy products (lacto-vegetarians) or dairy and eggs (lactoovo-vegetarians) (75). Approximately 2.5% of the entire US adult population (4.8 million people) report consumption of vegetarian diets, and approxi mately 1% report consuming vega n diets (75-77). There is an increasing trend for the younger segment of the popul ation to consume vegetarian diets (75). In a nationally-representative survey (75), the numbe r of self-defined vege tarians who reported no meat consumption was highest in the 20 to 29 y ear age group and was two to three times higher than that of 50 to 59 and 60 to 69 year old i ndividuals, respectively. This increasing trend in consumption of vegetarian diets is esp ecially prevalent among young adult women of

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26 reproductive age, documented by survey data in dicating that 20 to 25% of this group follow some type of vegeta rian diet (78). Multiple reports provide evidence that all ve getarians including lacto-vegetarians and lacto-ovo-vegetarians are at in creased risk for developing a B12 deficiency compared to omnivores (79-81), supporting the co nclusion that a vegetarian does not have to be a strict vegan for B12 status to be impaired (32, 81-85). Im paired B12 status may lead to elevated Hcy concentrations and increased risk for cardiovas cular disease, cancer and birth defect-affected pregnancies. A number of st udies indicate that vegetarians have significantly higher Hcy concentrations than omnivores a nd that the consumption of a vege tarian diet may be associated with elevated Hcy concentrations (59, 80, 86). For example, Mezzano et al. (59) reported that the Hcy concentration was 41% higher in vege tarians than in omnivor es and that Hcy was inversely related to serum B12 concentration. In a study compar ing B12 status of Taiwanese vegetarians and non-vegetarians, Hu ang et al. (87) reported that vegetarians had higher plasma Hcy concentrations than non-vegetarians (13.2 vs. 9.8 mol/L, respectively) and that serum B12 concentration was a strong pred ictor of plasma Hcy concentr ation. Another similar study conducted in a European population reported that Hcy concentration was significantly higher (11.6 mol/L) in a group of vegetarians compared to omnivorous controls (9.8 mol/L) and that the Hcy concentration increased as the vegetarian diet became more rest rictive, with vegans having the highest values (86). In the only study reported to date in which the B12 status of young adult vegans in the US has been evaluated, Carmel et al. (88) found that elevated Hcy concentration associated with dietary inade quacy of B12 was a major problem in young Asian Indian medical students with hyperhomocystein emia occurring in 25% of group. This study

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27 illustrates that well-educated young adults ar e among the vegetarians in the US whose consumption of a B12-deficient diets has been asso ciated with an elevation in Hcy concentration. Gene-Nutrient Interactions Transcobalamin 776C G The most common polymorphism affecting the TC B12 transport protein is a C G base substitution in DNA at base pair 776, which results in the substitution of pro line with arginine at codon 259 (74, 89). The estimated prevalence of the TC 776 CC, CG and GG genotypes are approximately 20%, 50%, and 30%, respectivel y (74, 89, 90). Our research group recently estimated the distribution (27% CC; 49% CG; 24% GG) of the TC 776 polymorphism in a large population group of young women for whom B12 stat us was previously reported, which is in agreement with previous studies (91). The potential influence of the TC 776 C G polymorphism on indices of B12 status has been investigated by several research groups (74, 89, 90). Afman et al. (30) found lower holoTC, total TC, and holo-TC/total-TC ratios in in dividuals with either the TC 776 GG or CG genotypes compared to those with the CC genotype. Miller et al. (89) reported a lower mean holo-TC concentration, a lower percent of to tal B12 bound to TC, and a higher mean MMA concentration in elderly subj ects with the TC 776 GG compar ed to the CC genotype. Our research group evaluated B12 status in young women with all three TC 776 C G genotypes (CC, CG, GG) (91). Mean holo-TC concentr ation was significantly lower in TC 776 GG compared to CC genotypes, and individuals with low (< 35 pmol/L) holo-TC had a significantly higher mean Hcy concentration. Al ternatively, some studies have reported no eff ect of the TC 776 C G polymorphism on B12 status or metabolism (92-94).

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28 Any reduction in B12 binding capacity, protein s ynthesis, or transport function caused by the TC 776 C G polymorphism could impair functional B 12 status, leading to reduced cellular availability of B12. If the polymorphism has a physiologically significant impact on B12 availability, it would likely be exacerbated by concurrently low B12 status due to insufficient dietary B12 intake. In a populati on where more individuals have ma rginal B12 status, such as in vegetarians, any negative effect of the TC 776 C G polymorphism would be expected to be more apparent. Malabsorption of Vitamin B12 The most common form of B12 mala bsorption is often termed food-bound malabsorption(95). Because only free B12 can bi nd to the transport prot eins and be taken up into the enterocyte or absorbed passively, any ph ysiological condition that reduces the ability to free B12 from the protein matrix will lead to malabsorption and can lead to a B12 deficiency. Approximately 5 to 25% of adults over the age of 60 y are estima ted to have some degree of food-bound B12 malabsorption due to an age-rela ted decrease in stomach acid, or achlorhydria (96). Because achlorhydria is so prevalent in older adults it is recommended that the daily requirement of B12 be met by consuming supplem ental forms of B12 (97). Vitamin B12 found in fortified foods and vitamin supplements is not protein bound and therefore, can be absorbed with normal efficiency even if gastric pH is high. Another less common, but often more severe form of B12 malabsorption, termed pernicious anemia, can occur in all age groups although incidence does in crease with age. Pernicious anemia is caused by a lack of IF re sulting from an autoimmune response, atrophy of the gastric mucosa, chronic gastriti s, and in rare cases a congenita l defect in the gene for IF. Congenital defects may lead to synthesis of an altered, and therefore non-f unctional IF protein or a complete lack of synthesis. Both conditions cause B12 deficiency at an early age and have

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29 been reported to be caused by a variety of geneti c mutations and post-translational defects. The autoimmune-based B12 malabsorption condition is more prevalent in older adults but has been observed in all age groups (98). In this case, the body recognizes either the IF itself or the gastric parietal cells as foreign and synthesizes antibodies to the protein or cell eliciting an immune response. Destruction of IF or the pari etal cells by this autoimmune response may occur to varying degrees resulting in variation in the severity of B12 malabsorption (98). Currently the only available di agnostic tests for pernicious anemia are not clinically practical. The Schilling test, wh ich involves ingestion of radio actively-labeled B12, a flushing dose of non-labeled B12, and collection of urin e over a period of 24 hours requires meticulous adherence to protocol making it erro r prone and costly (99-101). Pres ence of IF or parietal cell antibodies can be measured to diagnose pernicio us anemia; however, parietal cell antibodies can occur in other autoimmune diseases, and both te sts are only clinically meaningful in a subgroup of patients with autoimmune c onditions (102, 103). It has been hypothesized that changes in holo-TC in response to a supplemental dose of B 12 may be used to assess B12 absorption (28, 47, 104). Bor et al (20) reporte d a significant increase in holo-TC and TC saturation 24 and 48 hours after receiving three 9 g oral B12 doses. Since no blo od was collected before 24 hours (post baseline), the magnitude and pattern of ch ange of holo-TC during the first 24 hours could not be determined (47). In developing a clinic al diagnostic test, it is important to know the optimal time post dose at which to draw blood. Overall Rationale Vitamin B12 plays a central role in Hcy metabolism, and B12 deficiency has been associated with numerous health risks, including birth defect-affected pregnancies. Few studies have been designed to evaluate the relations hip between dietary excl usion or limitation of

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30 specific animal products and B12 status in indi viduals who do not take B12 supplements or consume B12-fortified foods. The proposed stud y will evaluate the association between B12 status and intake of specific animal-deriv ed food products among vegetarians who do not consume B12-containing supplements. Several studies, including one conducted by our research group (91), in dicate that the TC 776 GG genotype results in decreased holo-TC c oncentrations, and could therefore be a risk factor for a B12 deficiency. Although most st udies have not found a co rrelation between TC 776 C G genotype and Hcy or MMA, holo-TC and Hcy and MMA have been negatively correlated (89, 105). It is hypothesized that B12 transport, a nd thus metabolic function, may be impaired in individuals with the TC 776 GG ge notype, and that an effect on Hcy and MMA concentrations due to the TC 776 GG genotype may be evident in individuals with low B12 intake and status. These data could be used for public health screening and inte rvention approaches for adults whose combined dietary choices and genetic make -up may put them at higher risk for certain diseases or poor pregnancy outcomes. Inform ation generated from th is study could benefit individuals who exclude B12-dense food sources from their diets for reasons related to health or personal choice rather than religi on, culture, or the environment, as well as for individuals who consume strict vegan diets for religio us/cultural or environmental reasons. Hypothesis # 1 Moderate B12 deficiency will be more common in vegetarians not taking B12 supplements than in their omnivorous counterparts. Specific aim: To determine if non-supplement taking young adults who exclude animalbased foods and are not taking B12-containing su pplements are at a greater risk for a B12

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31 deficiency than those who eat animal-based foods by comparing B12 status indices between groups. Hypothesis #2 Vitamin B12 intake at the current RDA may not be sufficient to maintain normal B12 status. Specific aim: To determine the level of B12 intake associated with optimal status as defined by normal B12 status biomarkers. Hypothesis #3 Genotype status for the TC 776C G polymorphism will have a greater physiological impact on individuals with low B12 status th an those with normal B12 status. Specific aim: To determine if genotype status for the TC 776C G polymorphism further impairs B12 status in individuals with low B 12 intake by comparing B12 status indices among genotype groups in individuals with low and normal B12 status. Hypothesis #4 Holo-transcobalamin concentration can be used to assess B12 absorption. Specific aim: To determine if holo-TC concentration increases measurably in response to B12 supplementation within a 24 hour time period.

33 Figure 1-2 Overview of vitamin B12 (B12) ab sorption. (1) Food bound B12 is released in the acidic environment of the stomach. (2) Free B12 binds to haptocorrin and the complex travels to the duodenum. (3) Panc reatic proteases degrade HC. (4) Free B12 binds to intrinsic factor, wh ich is synthesized in the gastric parietal cells. (5) The B12 IF complex to travel to the ileum a nd is transferred across the ileal epithelium via receptor mediated endocytosis, along w ith 1% passive diffusion. (6) In the enterocyte, intrinsic factor is degraded by the lysosome. (7) Transcobalamin binds B12 at some point after releas e from intrinsic factor, this may occur in the enterocyte.

35 CHAPTER 2 VITAMIN B12 STATUS IS IMPAIRED IN A SUBGROUP OF HEALTHY YOUNG VEGETARIAN AND OMNIVOROUS ADULT MEN AND WOMEN Naturally occurring dietary sources of B12 are li mited to foods of animal origin, which if restricted in the diet may impair B12 status ( 40-42). Vitamin supplements and fortified foods can also contribute to B12 inta ke (97); however, it is estimated that ~70 % of the United States (US) population does not take supplements (106). Vegetarians, individual s who avoid some or all animal-derived foods, have lim ited dietary intake of B12 and may be at greatest risk for developing a B12 deficiency compared to omnivor es. Few data are avai lable on B12 status in young adult vegetarians in the United States, and further evaluation of B12 status in this subgroup of individuals is warrant ed to better determine relative risk of B12 deficiency and related disease. Clinical determination of B 12 deficiency relies on the availability of specific and reliable biomarkers of B12 status. Biomarkers currently us ed to assess B12 status include serum B12, MMA, Hcy and holo-TC concentrati ons. Although holo-TC is not yet used clinically, holo-TC is reported to be more sensitive than serum B12 and may be comparable to MMA as a biomarker of B12 status. The objectiv es of this study were to determine if young adult vegetarians who do not take B12 supplements are at a greater risk for B12 deficiency than omnivores not taking B12 supplements, and to comp are the various B12 status indicators within these groups. Subjects and Methods Subjects and Subject Recruitment Healthy adults (n = 388) from the Alachua county, FL community including university students, faculty and staff were recruited by flyers and newspaper advertisements with simultaneous recruitment for healthy adult vegeta rians and healthy adults. Subjects were screened by phone and selected based on the follow ing exclusion criteria: (a) < 18 y & > 49 y (b)

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36 major change in animal-product consumption (i.e. vegetarian or omnivore) habits during the past 3 years; (c) B12-containing supplement use with in 6 mo of screening; (d) chronic alcohol consumption (>1 drink/d of any kind); (e) use of tobacco products; (f) use of prescription medications other than oral contraceptives; (g) pe rsonal history of chroni c disease; (h) regular blood donations; and (i) pregna nt or lactating women. Potential subjects were asked about their meat consumption habits during the phone screening for initial classificati on as vegetarian or omnivore. Specifically subjects were asked How often do you consume (a) beef, (b) chicken, (c) turkey, (d) pork, and (e) fish. Subjects who responded never to all questions were tem porarily classified as vegetarian. This study was approved by the University of Florida Institu tional Review Board, and all subjects signed an informed consent prior to beginning the study. Study Design and Data Collection Between the hours of 7:00 am and 9:00 am s ubjects were scheduled for fasting blood sample collections. Subjects were called 24 hou rs prior to their scheduled appointment to remind them to fast overnight and the following morning. Following sample collection, subjects were given a small meal and a comprehensive in formation session explaining how to complete the National Cancer Institute Di et History Questio nnaire (DHQ). Subjects were asked to complete the questionnaire at home and return it within two weeks and to contact a designated member of our recent team personnel if they had any questions or problems completing or returning the questionnaire. For any unreturne d DHQs, individuals were contacted by phone or e-mail to determine if the questionnaires were lost in transit or if the subject had not had an opportunity to complete the DHQ instrument. S ubjects that chose not to return the DHQ were not included in the final data analysis. The DHQ has been validated for the estimation and

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37 quantification of dietary intake of all essential nutrients including B12 (107). Subjects were instructed to answer all questions based on their diet over the past 12 mo estimating the frequency of intake and portion size of 125 different food items. A total of 70 mL of blood were collected for analysis of the following indices: (a) serum holo-TC; (b) plasma B12; (c) serum MMA; (d) serum Hcy (e) serum folate, and (f) hematocrit. Sample Processing Blood samples were collected in ethylenedi aminetetraacetic acid (EDTA) and serum separator clot activator (SST) tubes. EDTA tubes were centrifuged for 30 min at 2000 x g at 4 C to separate and collect plasma for B12 analyses Serum separator tube s were centrifuged for 15 min at 650 x g at room temperature to separa te and collect serum fo r holo-TC, MMA, Hcy, and folate determination. All samples were stored frozen at 30 C until analysis. Competitive Binding Assays of Serum Holo-transcobalamin and Plasma B12. Serum holo-TC concentration was determ ined by radioimmunoassay (holo-TC RIA reagent kit; Axis Shield, Ulvenve ien, Oslo, Norway) based on the method of Ulleland et al. (45). Specifically, magnetic microspheres coated with anti-human TC monoclonal antibodies were incubated with each sample for a period of one hour to isolate both holo-TC and apo-TC. Once attached to the metal beads via antibody inter action, the TC protein a nd associated B12 were magnetically separated from the sample. Next, isolated TC was incubated with 57Co-labeled B12 tracer plus reducing agent followed by a denatu ring agent to free B12 from the TC protein. Finally, each sample was incubated with IF to which unlabeled and labeled B12 bind competitively based on their relative concentr ations. Remaining unbound B12 was removed and the relative radioactivity of each sample meas ured by gamma counter. Radioactivity of each

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38 sample in counts per minute (CPM) was compared to a standard curve with serum holo-TC concentration being inversel y associated with CPM. Plasma B12 was determined by RIA using a co mmercially available kit (Quantaphase II, Bio-Rad). Specifically, samples were incubated with a 57Co labeled B12 tracer in a 100oC water bath to convert all forms of B12 to cyanocobala min. Samples were brought to room temperature after boiling for 20 min, and then mixed with purified porcine IF bound to polymer beads and incubated for one hour. During in cubation labeled and unlabeled B12 compete for binding to IF at rates that match their relative concentra tions. Finally, samples were centrifuged, and supernatant containing unbound B12 was removed. Sample radioactivity was measured by gamma counter and B12 concentration was calcu lated using a standard curve on which the radioactivity was inversely re lated to B12 concentration. Measurement of Serum Homocysteine and Methylmalonic Acid. Serum Hcy and MMA concentrations were determined by gas chromatography mass spectrometry (Metabolite Laboratories, Inc. Denver, Colorado) (108, 109). Diet Analysis Daily B12 intake was assessed based on data obtained from the DHQ, which was modified to include additional B12-containing foods incl uding meats, mixed dishes, fortified foods and meat substitutes. The original DHQ is available online at http://appliedresearch.cancer.gov The DHQ was scanned by Optimal Solutions Corporati on (OSC), Lynbrook, New York. Dietary data obtained from scanning the questionn aires was sent to the Universi ty of North Carolina, Chapel Hill as an ASCII text file, and then analyzed using the Diet*Calc Analysis program. In order to analyze the modified questionnaire, the Diet*C alc program was updated to include nutrient values for all of the food items added based on data from the United States Department of

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39 Agriculture (USDA) National Nu trient Database for Standard Reference and nutrition label information when data was not available from the former USDA database Diet*Calc is a freeware program and can be downloaded from the National Cancer Institute website (www.riskfactor.cancer.gov ). Subject Dietary Intake Classification Individuals were classified as vegetarian or omnivore based on their responses to the DHQ. Individuals classified as vege tarians were those who reporte d no consumption of any meat products (i.e., beef, poultry, pork, lamb and seaf ood) or meat-based mixed dishes and who reported consuming dairy products and eggs ne ver to daily. Omnivores were defined as individuals who consumed any meat, poultry or s eafood products. In addition to the preexisting questions in the DHQ that asked about all types of meat consumption, a new question was added to more accurately classify subjects into specif ic dietary intake categories. This question required subjects to indicate the foods they ne ver consumed including (a ) beef, (b) chicken, (c) turkey, (d) pork, (e) fish, (f) dairy products, and (g) eggs. Statistical Methods. Vitamin B12 status based on the measured B 12 indicators and dietary B12 intake were compared between groups using analysis of variance (ANOVA) with an alpha of 0.05. Dependent variables also were classified as n ormal vs. abnormal according to whether they were above or below esta blished thresholds (B12, 148 pmol/L; holo-TC, 35 pmol/L; MMA 271 nmol/L; and Hcy 12 mol/L) and comparisons with respect to dietary group were done by Pearson Chi-Square tests. The distributions of all possible combinations of normal and abnormal test results were calculated and the rate of each B12 indicator being abnormal when all others were normal or abnormal was compared using a Chi-square test. Age distributions were

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40 compared between the two dietary groups by ANOV A, while race and gender for the two dietary groups were compared by Pearson Chi-Square tests. Results One hundred and twenty one vegetarians and 18 1 omnivores completed the study (total n = 305). Of the 388 enrolled subjects, 62 were ex cluded due to reporting supplement use, and 23 did not complete the DHQ. All results are reporte d as mean SD unless otherwise noted. There was a significant difference in age and BMI betw een groups, with vegetarians being older and having a lower BMI. There were no significant di fferences in gender or ethnicity between diet categories (Table 2-1). Total B12 intake (g/d SD) and B12 in take expressed as g/1000 kcals were significantly lower (P < 0.001) in vegetarians than in omnivore s (Table 2-2). Plasma B12 concentration was significantly lower (P < 0.01) in vegetarians than omni vores (Table 2-2). Serum MMA concentration was significantly highe r (P = 0.001) in vegetarians compared to omnivores (Table 2-2). Mean holo-TC and Hcy concentrations were not significantly different between groups (Table 2-2). Vitamin B12 deficiency, based on having a valu e outside the normal range for one or more of the B12 status indicators, was twice as prev alent (P < 0.001) in vege tarians than omnivores (42% and 23%, respectively). Impaired B12 status based on concentrations of plasma B12, serum holo-TC and serum MMA combined also was significantly greater in vegetarians compared to omnivores. Specifically, more than twice as many (P < 0.05) vegetarians had low serum holo-TC (< 35 pmol/L), plasma B12 (< 148 pmol/L), and elevated serum MMA (> 270 nmol/L) concentrations than omnivores (Figure 2-1). There was no significant difference in the percentage of vegetarians versus omnivores with elevated Hcy (> 12 mol/L).

PAGE 41

41 Subjects were cross-tabulated by B12 status as defined by having a value within (+) or outside (-) the normal range for B12, holo-TC and MMA singly and in combination (Table 2-3). Because of the small numbers of subjects within each of the resulting 8 categories, statistical analysis was not done; however, the likelihood of one B12 status indicator being abnormal while the remaining tests were normal was conducted. In this analysis, B12, holo-TC and MMA were more likely to be abnormal when one or more of the other indicators were abnormal (23, 38, and 36 % of the time, respectively) compared to wh en all others were normal (6, 11, 10 % of the time, respectively) (Figure 2-2). Discussion The primary objective of this study was to asse ss and compare the B12 st atus and intake of young adult vegetarians and omnivores who do not take B12-containing vitamin supplements to determine if vegetarians are at greater risk for developing a B12 deficiency than their omnivorous counterparts. Although long term a dherence to a vegetarian diet can provide substantial health benefits (110), limitation of most or all animal -based foods, particularly when B12-fortified foods or supplements are not added to the diet, can increase the risk for developing a B12 deficiency. It has been estimated that B12 intake in the general US population is adequate (111), however, data from this study suggest that a subgroup of healthy young, non-supplement using adults may not be consuming adequate B12. Within the vegetarian groups, 43% were determined to be potentially B12 deficient base d on having a value outside the normal range for one or more B12 status indicators, 61% of whom had elevated MMA, indicating metabolic impairment. Surprisingly, of the omnivores, 23% were potentially B12 deficient, with 48% of them having an elevated MMA concentration, su ggesting that even a m eat-containing diet may not provide sufficient B12.

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42 This is of particular concern for young wome n of childbearing age, who might chose to avoid meat in an attempt to lower fat and choles terol intake, but do not consume other sources of B12. Nutrient availability is crucial in the fi rst 180 days of pregnanc y, during a portion of which a woman might not even know she is pregnant (112). This issue has been addressed in relation to folic acid; however unlike folic acid, it is no t well recognized that a B12 deficiency is an independent risk factor for ne ural tube defects (57, 113, 114). The DHQ used to assess dietary B12 intake asks subjects to recall di etary intake over the past 12 mo and answer questions based on their be st estimate of food intake. Full analysis of DHQ data was the focus of an investigation c onducted by another member of the laboratory group and will be reported separately. The data obtained from the analyses conducted for this study were used to group individual s within a similar range of overa ll B12 intake and to identify foods eaten or excluded by each subject. This allo wed for a very strict definition of vegetarian subjects who reported no meat consumption and omnivore subjects who reported some degree of meat consumption. Previous studies, the majo rity of which were conducted in Europe, used a similar approach to classify individuals as vege tarian. Vegetarians have previously been subgrouped into lacto-ovo-vegetarians, lacto-vegetarian and vegan cat egories, with the greatest deficiency associated with the vegan diet relative to other vegetarian sub-groups (81). It is widely believed in the US, that a B12 deficien cy is not a problem in healthy young adults who consume at least some animal-based products. The data from the present study do not support this perception. In addition to the moderate deficiency that was detected in the ve getarian and omnivorous groups, a small subset (including bo th omnivores and vegetarians) was determined to be severely B12 deficient as evidenced by reports of ne urological problems in cluding numbness and a

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43 tingling sensation in the extremities. Of thes e subjects none had previously sought out medical attention for these symptoms, which illustrates that B12 deficiencies may go undetected among seemingly healthy young adult men and women. Earl y detection of a B12 deficiency is the most effective way to prevent progressi on to serious health complications Recent studies suggest that measurement of holo-TC is superior to serum B1 2 because only holo-TC is taken up into cells, and therefore only that portion ( 20%) is biologically active (115). Addi tionally, holo-TC has been reported to be more sensitive to changes in B12 intake than total serum B12 (28). Miller et al. (116) reported that the use of holo-TC and serum B12 together as a ratio may be superior to the use of either alone. Their data suggest ( 116) that use of combined holo-TC and serum B12 measurement could lead to thr ee possible diagnoses; normal, po ssible deficiency (only 1 low indicator), and deficient (both indicators low). Finally MMA is still considered by many researchers to be the gold standard (54, 117). In the current study, we cross tabulated subjects by B12 status as defined by having a value within or outside the normal range for B12, holo-TC and MMA singly and in combination with each other. Statistical analysis co uld not be conducted due to limited sample size; however, a combined analys is of the two diet grou ps indicated that it is more likely that when the value of one biomarke r is outside the normal range at least one other indicator is more likely to also be outside the normal range. A dditionally, the length of time an individual adheres to a B12-in sufficient diet will have diffe rential effects on specific B12 biomarkers (32). Considering the differences in the primary biomarkers of B12 status, holo-TC may be initially affected followed by a decrease in serum B12 (once B12 stores have been depleted), and finally an elev ation in MMA indicating impaired cellular B12-dependant enzyme function. The data from the current study do not definitively support one B12 biomarker as

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44 being superior to another; however, in a clinical setting the more biomarke rs that are outside the normal range, the more likely a B12 deficiency exists. In conclusion, the high incidence of impaired B12 status observed in these otherwise healthy young adults was unexpected. These data i ndicate that dietary in take alone may not be meeting the B12 needs of non-supplement using adults especially vegetarians. Further research focusing on B12 status and inta ke in individuals consuming both vegetarian and low-meat containing diets is warranted. Assessment of B12 status by a combination of biomarkers may provide a more definitive diagnostic approach prior to treatment. Table 2-1 Characteristics of study groups Vegetarians (n = 121) Omnivores (n = 181) Age (y; mean SD) 28 9b 24 6 BMIa (mean SD) 22.9 3.9b 23.9 4.1 Gender (count) Female 67 98 Male 54 83 Race/Ethnicity (count) White 69 119 African American 6 11 Asian 17 17 Asian Indian 12 4 Hispanic 12 26 Other 5 4a Body mass index (BMI); b Significantly different from omnivores (P < 0.05)(ANOVA)

47 CHAPTER 3 VITAMIN B12 INTAKE AT THE CURR ENT RDA LEVEL IS NOT OPTIMAL The current RDA for B12 was established ba sed on data from patients who were being treated for pernicious anemia. Specifically, the am ount of B12 required in an injectable form to normalize serum B12 in patients di agnosed with pernicious anemia was determined to be the daily B12 requirement fro adults. The B12 RDA (2.4 g/d) was derived by adjusting the estimated B12 requirement for bioavailability, ente rohepatic recirculation, and the CV for 97 to 98 % of the population. It has been suggested that the RDA for B 12 is not optimal, and that B12 status is improved with intakes up to 6 g/d (118, 119). The object ive of this analysis was to determine the level of dietary B 12 intake associated with optimal B12 status as defined by B12 status biomarkers within the normal range. Subjects and Methods Subjects and Subject Recruitment Healthy adults (n = 302) were recruited from the Alachua county, FL community including university students, faculty and staff. Specifical ly, subjects were screened by phone and selected based on the following inclusion cr iteria: (a) 18 to 49 y (b) no change in meat consumption habits over the past 3 years; (c) no B12-containing supplement use within the past 6 mo; (d) limited chronic alcohol consumption (<1 drink/d of any kind); (e) no use of tobacco products; (f) no chronic use of prescription medications other than oral contraceptive agen ts; (g) no history of chronic disease; (h) no chronic blood donations; a nd (i) non-pregnant and n on-lactating. All 302 qualified subjects from the first pa rt of the current study were in cluded in this analysis. The approved institutional review board informed consent form signed by the subjects at the beginning of the study included cons ent for all aspects of studies described in the manuscript. Study Design and Data Collection

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48 Subjects were called the day before their sc heduled study day to remind them to fast overnight (8 hours) and the following morning prior to having their blood drawn. Between the hours of 7:00 am and 9:00 am qualified subjects were scheduled for blood sample collection followed by a comprehensive information session explaining how to complete the National Cancer Institute Diet History Qu estionnaire (DHQ) used to assess dietary intake. A total of 70 mL of blood were collected for analysis of the following indices: (a) serum holo-TC; (b) plasma B12; (c) serum MMA; (d) serum homocysteine (H cy) (e) serum folate, and (f) hematocrit. Sample Processing and Analysis Blood samples were collected in EDTA and SST clot activator tubes. EDTA tubes were centrifuged at 2000 x g at 4 C for 30 min to obtain plasma for B12 analyses. SST tubes were centrifuged at 650 x g at room temperature for 15 min to obtain serum fo r determination of holoTC, MMA, Hcy, and folate concentra tions. Samples were stored at 30 C until analysis. Serum holo-TC concentration was determined by radi oimmunoassay (holo-TC RIA reagent kit; Axis Shield, Ulvenveien, Oslo, Norway) based on the me thod of Ulleland et al (45) using magnetic microspheres coated with anti-transcobalamin m onoclonal antibodies to is olate both holo-TC and apo-TC, and 57Co-labeled B12 as a tracer. Plasma B12 concentration was determined by RIA using a commercially available kit (Quanta phase II, Bio-Rad). Serum Hcy and MMA concentrations were determined by gas ch romatography mass spectrometry(Metabolite Laboratories, Inc. Denver, Colorado) (108, 109). Diet Analysis Daily B12 intake was estimated based on data obtained from the DHQ, which was modified to include an extensiv e list of B12-containing foods including meat containing mixed dishes, fortified foods, and meat substitutes. The unmodified DHQ is available for review online

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49 at http://appliedresearch .cancer.gov. The DHQ was scanned by Optimal Solutions Corporation (OSC), Lynbrook, New York. Once scanned OSC sent the dietary data as an ASCII text file to the University of North Carolina, Chapel Hill (UNC), where the data were analyzed using the Diet*Calc Analysis program modified for this ve rsion of the DHQ. This freeware program can be downloaded from the NCI website (www.riskfactor.cancer.gov). Th e B12 content of the food items added to the DHQ was obtained from the US DA National Nutrient Database for Standard Reference and nutritional labels (39). Statistical Analysis Results are reported in the text as mean SD with an alpha = 0.05. The dependent variables B12, holo-TC and MMA c oncentrations were classified as normal vs. abnormal according to falling above or below an established threshold (B12, 148 pmol/L; holo-TC, 35 pmol/L; MMA 271 nmol/L; and Hcy 12 mol/L) and comparisons with respect to dietary B12 intake were performed using the Pearson Chi-Squa re test. Subjects were divided into dietary B12 intake quintiles, and B12 status base d on plasma B12, serum holo-TC, MMA, and Hcy concentrations were compared between groups using ANOVA with an alpha of 0.05. The Least Significant Difference (LSD) method of multiple comparisons was used for assessment of differences between quintiles. The LSD ensure s every target population paired difference in means will be within +/LSD of the corres ponding difference in sample means with 95% confidence. The data were analyzed usi ng EXCEL (Microsoft, Redmond, WA) and PRISM software (Graph-Pad Software Inc. El Camino, CA). Results Three hundred and two healthy you ng adult (18 to 48 y) men and women were included in this analysis. Subject characteristics are listed in Table 3-1. Seventy si x subjects (25 %) had an intake below the RDA of 2.4 g/d. Dietary B12 intake was si gnificantly correlated (P < 0.05)

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50 with B12, Hcy, and MMA concentr ations, but not with holo-TC concentration (Table 3-2). Individuals with low plasma B12 (< 148 pmol/L) or holo-TC (< 35 pmol/L) concentrations and those with elevated serum MMA (> 270 nmol/L) or Hcy (> 12 mol/L) concentrations had significantly lower dietary B12 in take than those with normal concentrations (Figure 3-1). To further evaluate the influence of B12 inta ke on B12 status, subjects were ranked and grouped by quintile of B12 intake. The mean co ncentration for each B12 status indicator was plotted against the mean B12 intake for each quintile group (Figure 3-2). Mean holo-TC, B12, MMA and Hcy concentrations were significantly different (P < 0.05) among B12 intake quintile groups (Figure 3-1). Specifically, mean holo-TC increased (P < 0.01) from quintile 1 through 3 and then maintained approximately the same value from quintiles 3 through 5, which was associated with a mean B12 intake of 4.3 g/d. Mean plasma B12 concentration increased (P < 0.001) from quintile 1 through 4, reaching a plat eau from quintile 4 through 5 corresponding to a B12 intake 6.7 g/d. Mean MMA decreased (P < 0.001) from quintile 1 through 2 then reached a plateau, which was associated with a B12 intake of 2.7 g/d. Homocysteine changed to the smallest degree, but decreased (P < 0.05) from quintile 1 through 3 maintaining this approximate value thought quintile 5, which corresponded to a B12 intake > 4.3 g/d. In the case of holo-TC, B12, and Hcy concentr ations, the means acros s all quintiles of B12 intake were in the normal range ; mean MMA concentration was elev ated in quintile 1 and within the normal range for all subsequent quintiles. Th e proportion of subjects with B12 deficiency as defined by abnormal biomarkers within each quin tile group (i.e. low B12, low holo-TC, elevated MMA or elevated Hcy) decreased significantly from the lowest to highest B12 intake quintile for all indices measured (Table 33). Specifically, in the group of subjects who consumed > 3.4 g/d of B12 there was a signifi cantly smaller percentage of subjects with low holo-TC or

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51 elevated MMA concentrations than those consuming 3.4 g/d. In the group of subjects who consumed at least the RDA for B12, there was a significantly lower number of individuals with a low plasma B12 concentration than in the group of individuals who cons umed less than the RDA (Table 3-3). Discussion In this study, the relationship between estim ated B12 intake and a panel of B12 status biomarkers were assessed in order to evaluate the adequacy of the current RDA for B12. It has been suggested by another research group that a B12 intake of 6 g/d was associated with improved concentrations of all B12 biom arkers compared to an intake of 2.4 g/d (118). Although the findings in the current study vary de pending on the specific biomarker, the data indicate that an intake greater than the current RDA is associ ated with normal B12 status. Overall, no clear conclusion can be drawn from these data as to a specific intake level of B12 that might result in normaliza tion of all B12 biomarkers; howev er, the data suggest that the current RDA may not be optimal. In the first investigation of this study group, subjects were classified as vegetarian or om nivore, and inadequate B12 status was detected in a surprising number of individuals in both groups. Specifi cally, 40% of vegetarian s and 11% of omnivores were determined to have abnormal values for on e or more of the four B12 biomarkers. The mean B12 intake of both groups exceeded the RDA. In the current analysis, the third quintile corresponded best to the current RDA for B12 with a mean and range of B12 intake at 2.7 g/d and 2.0 to 3.4 g B12, respectively. Beyond the second qui ntile, the mean concentrations of holo-TC and B12 increased; mean Hcy decrease d and overall rate of deficiency decreased significantly. This suggests that B12 status improves with inta kes above the second quintile,

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52 which in this study was represen ted as an intake level of 3.4 g/d. Little or no change was observed in subsequent qui ntiles, suggesting that an intake level above 3 g/d may be required. One limiting factor of the current study is the use of a FFQ to estimat e B12 intake rather than a 7-day weighed food record as was used in the study by Bor et al. (118). Because the DHQ relies on subject recall and estimation of intake over the past 12 mo, it is more prone to error and less precise than a direct measure. In additi on, neither the one week weighed food record nor a FFQ gives an estimate of duration of a particular diet, and because B12 status is slow to change relative to changes in B12 intake, estimated inta ke over one week or even over one year may not always correlate well with status at a given time. In a very large study using data of from the Framingham Offspring population, which also used an FFQ to assess B12 intake, improvements in B12 status were observed for intakes up to 10 g/d (119). Therefore, data from the current study in addition to that from two previous stud ies agree with the conclusion that the RDA for B12 is inadequate to maintain normal B12 st atus (39, 118). Further i nvestigations focusing specifically on changes in B12 status with increas ing B12 intake are warranted to address this issue and derive an estimate of B12 intake that is consistent with maintenance of normal B12 status. Because the current R DA was established using data from research conducted with patients who had pernicious anemia and who were injected with B12 rather than in healthy individuals consuming dietary B 12, there is clear just ification for conducting controlled feeding studies to obtain pertinent data necessary to re vise the current RDA. The RDAs are not intended to be therapeutic recommendations for individual s with disease conditions such as pernicious anemia. Recommended intake of B12 for the adu lt population should apply to a majority of the population, potentially with additio nal recommendations for some sub-groups such as vegetarian

57 CHAPTER 4 GENOTYPE FOR THE TRANSCOBALAMIN 776C G POLYMORPHISM IS NOT ASSOCIATED WITH ABNORMAL VITAMIN B12 STATUS BIOMARKERS IN HEALTHY ADULTS Transcobalamin (TC), the B12 transport protein required for cellular uptake is essential to maintain B12 metabolic function (26, 120). A common genetic polymorphism for TC (TC 776 C G) may impair the metabolic ro le of this protein (74, 89). It is hypothesized that B12 transport and thus metabolic function will be impaired in individuals with the homozygous variant genotype (GG) for the TC 776 C G polymorphism. The metabolic and health-related risks associated with this polymorphism are pred icted to be exacerbated by the consumption of low-B12 vegetarian diets that ex clude specific animal-d erived foods. The primary goals of this study were to evaluate the effects of the TC 776 C G polymorphism on B12 metabolism in young adult men and women who consume a low B12 diet compared to those consuming adequate B12. Subjects and Methods Subjects and Subject Recruitment Healthy adults (n = 302) were recruited from the Alachua county, FL community including university students, faculty and staff. Subjects we re initially screened by phone and selected for the study based on the following inclusion criteria: (a) 18 y & 49 y (b) no change in meat consumption habits during the last 3; (c) no B12 containing supplement use within the past 6 mo; (d) limited chronic alcohol consumption (<1 drin k/d of any kind); (e) no use of tobacco products; (f) no chronic use of prescription medications other than oral cont raceptive agents; (g) no history of chronic disease; (h) no ch ronic blood donations; and (i) nonpregnant and non-lactating. All subjects from the first part of the research described in the manuscript were included in this

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58 analysis after a second informed consent form ap proved by the University of Florida Institutional Review Board that was specific for genetic analysis. Study Design and Data Collection Subjects were called the day before their sc heduled study day to remind them to fast overnight (8 hours) and the following morning prior to having their blood drawn. Qualified subjects were scheduled for fasting blood sample collection to be perf ormed between the hours of 7:00 am and 9:00 am followed by a comprehens ive information session explaining how to complete the National Cancer Institute Diet Hi story Questionnaire (DHQ), which was used to assess dietary intake. A total of 70 mL of blood were collected for analysis of serum holo-TC; plasma B12; serum MMA; serum homocysteine (Hcy), serum folate, and DNA extraction. Sample Processing and Analysis Blood samples were collected in EDTA and SST clot activator tubes. EDTA tubes were centrifuged at 2000 x g at 4 C for 30 min to obtain plasma for B12 analyses. SST tubes were centrifuged at 650 x g at room temperature for 15 min to obtain seru m for holo-TC, MMA, Hcy, and folate determination. Samples were stored at 30 C until analysis. Serum holo-TC was determined by radioimmunoassay (holo-TC RIA reagent kit; Axis Shield, Ulvenveien, Oslo, Norway) based on the method of Ulleland et al. (45) using magnetic microspheres coated with anti-transcobalamin monoclonal antibodies to isolate both holo-TC and apo-TC, and 57Colabeled B12 as a tracer. Plasma B12 was dete rmined by RIA using a commercially available kit (Quantaphase II, Bio-Rad). Serum Hcy and MMA concentrations were determined by gas chromatography mass spectrometry (108, 109).

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59 Genotype Determination DNA was extracted from blood as previously described (121) using a commercial kit (Quantum Prep, BioRad, Hercules, CA) and stan dard laboratory procedures. Genotypes of potential subjects were determined using D ynamic Allele Specific Hybridization (DASH) performed by DynaMetrix (Stockholm, Sweden ). Briefly, a short PCR product was created spanning the polymorphic position. One PCR primer was 5'-labeled with biotin for attachment of the amplified targets to streptavidin-coated 96-well microtiter plates. Following denaturation and a wash to remove the unbound strand, an alle le-specific probe was hybridized to the bound target DNA strand at low temperat ure in the presence of the doublestrand specific intercalating dye Sybr Green. Finally, the temperature was st eadily increased while recording the probe-target duplex melting temperature, as monitored by di minution of Sybr Green fluorescence with a quantitative PCR analysis device. Properly designed matched target-probe duplex es have higher melting temperatures than those with single-base mismatches, enabling una mbiguous allele discrimination. Heterozygous samples show two separate phases of denaturatio n. For analysis, the negative derivatives of the melting curves are plotted. A single peak at a lower temperature indica tes homozygous allelic mismatch to the probe, and a single peak at a higher temperature, a homozygous match. A double peak is generated from a he terozygous sample (Figure 4-1). Diet Analysis Daily B12 intake was estimated based on data obtained from the DHQ, which was modified to be inclusive of an extensive list of B12-containing foods in cluding meat containing mixed dishes, fortified foods, and meat substitute s. The unmodified DHQ is available for review online at http://appliedresearch.cancer.gov. The DHQ was scanned by Optimal Solutions Corporation (OSC), Lynbrook, New York. Once s canned, OSC sent the di etary data as an

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60 ASCII text file to the University of North Ca rolina, Chapel Hill (UNC). Data was analyzed using the Diet*Calc Analysis pr ogram modified for this version of the DHQ. This freeware program can be downloaded from the NCI website (www.riskfactor.cancer.gov). The B12 content of foods added to the DHQ were base d on up-to-date information from the USDA National Nutrient Database for Standard Reference and nutriti on labels (39). Statistical Methods Results are reported in the text as mean SD with an alpha = 0.05. Vitamin B12 status, based on measurement of plasma B12, serum hol o-TC, the ratio of holo-TC to B12, MMA, and Hcy, was compared among genotype groups with and without an adjustment for B12 intake, using ANOVA with an alpha of 0.05. Dependent vari ables also were classi fied as normal vs. abnormal according to falling above or below an established threshold (B12 148 pmol/L; holo-TC 35 pmol/L; MMA 271 nmol/L; and Hcy 12 mol/L) and comparisons with respect to genotype were performe d using a Pearson Chi-Square test Qualitative data including gender, age, and ethnicity were compared usi ng a Pearson Chi-Square test. The data were analyzed using Statistical Analysis System Soft ware (SAS Institute Inc. Cary, NC) and PRISM software (Graph-Pad Software Inc. El Camino, CA). Results No significant differences were detected among genotype groups for gender, age, or BMI, however there were differences in ethnic distri bution (Table 4-1). There were no significant differences among genotype groups with or without adjustment for B12 intake, for holo-TC, MMA, and Hcy concentration whether considered al one (Table 4-2) or in combination with low B12 status (Table 4-3). There was no signi ficant difference in B 12 among genotype groups, though there was a trend for higher B12 in th e TC 776 GG group (Table 4-3; P < 0.01). In addition, there were no significant differen ces among genotype groups in the number of

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61 individuals with values outside the normal range for plasma B12, holo-TC, MMA, or Hcy (Table 4-4). Individuals with the TC 776 GG genotype ha d a significantly lower (P < 0.05) ratio of holo-TC to plasma B12 than individuals with the CC genotype (Table 4-2). Total TC was significantly lower (P < 0.001) in the TC 776 GG genotype group co mpared to both the CG and CC genotype groups (Table 4-2). Transcobalami n saturation was not significantly different among groups. Discussion Studies investigating the effect of the TC 776 C G polymorphism have resulted in conflicting findings. In a previ ous study by our laboratory, significan t differences were detected in holo-TC concentration among the TC 776 genot ype groups, however in the present larger study, which included a wider range of B12 intake by subjects, a significant difference was not detected. In addition, there we re no significant differences in Hcy or MMA concentration among the genotype groups, further suggesting no real phys iological impact of this single base pair mutation of the TC gene on bioche mical indexes of B12 metabolism. Even when considering the combined influence of the polymorphism and low B12 status, there were no significant differences in any B12 status biomarkers among the genotype groups, indicating no effect on B12 metabolism in B12 impaired individuals. Interestingly, some significant differences were found among genotype groups, suggesting a moderate effect of the polymorphism on TC pr otein synthesis or catabolism. Specifically, total-TC concentration was lo wer in subjects with the TC 776 GG genotype. Transcobalamin saturation, however, was not different among ge notype groups, suggesting no effect on the ability of TC to bind B12. The ratio of holo-TC to B12 also was significantly lower in subjects with the TC 776 GG genotype compared to the CC genotype, though there was no significant

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62 difference in mean holo-TC among the groups. B ecause there was a significant difference in total-TC among genotype groups but not in TC satu ration there must have been some difference in holo-TC as well, because TC saturation is the ratio of holo-TC to total-TC. Although the differences were not significan t, holo-TC was somewhat lower and TC saturation somewhat higher in subjects with the TC 776 GG genotype compared to the CC genotype. Additionally, because there was no difference in MMA con centration among genotype groups, even in combination with low B12 status, the reduced concentration of total-TC in the TC 776 CC genotype likely has no important physiological effect on B12 me tabolism or functional status. Previous studies focusing on other higher risk groups, such as the in dividuals with low B12 intake included in this study, also have not detected a significant effect of the TC 776 C G polymorphism (92, 93, 122). Fodinger et al (93) reporte d no significant differe nce in holo-TC or Hcy concentration in end-stage renal disease patients with the TC 776 GG or CC genotypes. Wans et al (92) compared holo-TC, B12, Hcy a nd MMA concentrations in elderly subjects with the TC 776 CC and GG genotypes, and reported a lo wer holo-TC concentration in subjects with the TC 776 GG genotype compared to the CC genotype, but no differe nce in B12 or MMA concentrations. Comparing the absolute difference in mean values in the study by Wans et al. to the current study, the differences were 4 pmo l/L versus 51 pmol/L, respectively, for B12 (GG mean CC mean); and 22 pmol/L versus 6 pmol/L respectively for holo-TC (GG mean CC mean). Differences in initial B12 status coul d account for the discrepa ncies seen in the many studies examining the relationship betw een genotype status for the TC 776 C G polymorphism and B12 status. Because such small differences in B12 status may overcome any negative effect of the polymorphism, and because changes in metabo lic indicators of B12 st atus such as Hcy and

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63 MMA are not consistently observed, it is unlikely that any B12-re lated metabolic change related to this polymorphism is of clinical concern. It is important to note that the developing embr yo may be at risk of negative consequences of metabolic changes associat ed with genetic polymorphisms that coexist with suboptimal nutrient intake. A polymorphism affecting a ke y folate enzyme, met hylenetetrahydrofolate reductase (MTHFR 776 C T), is associated with a significant increase in risk for neural tube defects, and the risk is exacerbated when folate intake is low (123-125). Again, reports of the effect of the TC 776 C G polymorphism on birth defect ri sk have been mixed (30, 94, 126, 127), but most results suggesti ng an increased risk for preg nant women with the TC 776 GG genotype are not definitive. Potentially, combin ed effects of several polymorphisms that might interfere with B12 absorption or metabolism c ould be physiologically important and further investigation may be warranted based on findings from recent studies that considered several types of birth defects (128-130). A) TC 776 C G Figure 4-1 Melting curve plots for Dynamic A llele Specific Hybridization analysis of polymorphism the TC 776C G polymorphism. Negative de rivatives of Sybr Green fluorescence vs. time plots are shown for tw o samples of each allele combination. Single peak at a lower temperature ( ) indicates homozygous allelic mismatch to the preferred probe; single peak at a higher temperature ( ), a homozygous match; double peaks, a heterozygous sample.

66 CHAPTER 5 HOLO-TRANSCOBALAMIN IS AN INDICA TOR OF VITAMIN B12 ABSORPTION IN HEALTHY ADULTS WITH NORMAL VITAMIN B12 STATUS Circulating B12 is bound to one of two ca rrier proteins, haptocorrin (HC) or transcobalamin (TC). Although the majority of B12 (~80%) is bound to HC (holo-HC), only TC bound B12 (holo-TC) can be taken up by body cells (26). Depletion of total body B12 occurs slowly, and is often a result of malabsorption, which is difficult to di agnose clinically (13, 81, 131, 132). Currently the only ava ilable diagnostic tests for v itamin B12 absorption are not clinically practical. It has been hypothesized that changes in holo-TC in response to a supplemental dose of orally administered B12 may be used to assess B12 absorption (28, 47, 104). Bor et al (20) reported a significant increase in holo-TC and TC saturation 24 and 48 hours after receiving three 9 g oral B12 doses. Since no blood was collected before 24 hours (post baseline), the magnitude and pattern of change of holo-TC during the first 24 hours could not be determined (47). In developing a clinical diagnos tic test, it is important to know the optimal time post dose at which to draw blood. The objective of this study was to evaluate the post-absorption response of holo-TC to oral B12 relativ e to other indicators of B12 status. Subjects and Methods Subjects Twenty one healthy adult men (n = 13) a nd women (n = 8) (18 to 49 y) from the Gainesville, Florida community were selected ba sed on the following inclusion criteria: (a) serum B12 concentration > 350 pmol/L at time of screening; (b) no B 12-containing supplement use or B12 injections during past year; (c) no us e of tobacco products; (d) no history of chronic disease; (e) non-pregnant and nonlactating; (f) non-anemic (Hgb 11 g/dL [7.4 mmol/L], females; 12 g/dL, [8.1 mmol/L] males); (g) normal blood chemistry profile; (h) BMI between 18 and 29; and (i) no blood donations within 30 days of the study.

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67 Study Design and Data Collection All participants signed an informed consent form approved by the Un iversity of Florida Institutional Review Board prio r to the initiation of the study. Individuals had a fasting blood sample drawn at the University of Florida Shands General Clinical Resear ch Center (GCRC). Subjects heights and weight s were measured and a medi cal history questionnaire was completed. Blood analyses included serum B12, bl ood chemistry profile, hematological indices, and a pregnancy test for women. Eligible subjects were admitted to the GCRC th e evening before (day 0) the intervention. The following morning (day 1) after an overnight fa st, an indwelling catheter was inserted for all blood collections during day 1. Blood samples were collected a total of 17 times starting on day 1 through day 3, and three 9 g B12 doses were orally administ ered at six hour intervals on day 1 beginning after the baseline blood draw (Figure 1). Immediately after taking each B12 dose, subjects consumed a piece of bread and 236 ml (8 oz) of juice to improve absorption efficiency. In addition to the bread and juice consumed w ith each B12 dose, subjects were given a midmorning snack and lunch at 2 hours and 3.5 hours, respectively after dose 1. Dinner was fed 4 hours after dose 2, and an evening snack was pr ovided 3 hours after dose 3. The RDA for B12 was provided in the diet on day 1 and on day 2. Take-home meals were provided on day 2 of the study. Water and non-caffeinated, non-caloric beve rages were allowed ad libitum. Subjects remained in the GCRC overnight and were allowe d to leave on pass following the collection of a fasting blood sample the morning of day 2. Subj ects returned on the morning of day 3 at which time a final fasting blood sample was drawn. Biochemical Analysis At each blood collection, holo-TC, total-TC, B 12, and plasma albumin concentrations were determined. The ratios of holo-TC concentratio n to total-TC concentration (TC saturation) and

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68 holo-TC concentration to B12 concentration (holo-TC/B12) we re determined to assess changes in these indicators in relation to one anothe r. Additionally methylmalonic acid (MMA), creatinine, serum folate, and homocysteine (Hcy) c oncentrations were measured at baseline. The B12 supplement (9 g cyanocobalamin) was prepared by West lab Pharmacy (Gainesville, FL). The B12 content of the supplement was validated by an independent laboratory (Analytical Research Laboratories, Oklahoma City, OK). Sample Processing and Analysis Blood samples were collected in EDTA and SST clot activator tubes. EDTA tubes were centrifuged at 2000 x g at 4 C for 30 min to obtain plasma for B12 analyses. SST tubes were centrifuged at 650 x g at room temperature for 15 min to obtain seru m for holo-TC, MMA, Hcy, and folate determination. Samples were stored at 80 C in the GCRC until analysis. Serum B12 and folate concentrations were assayed on the Advia Centaur automated immunoassay system (Bayer A/S, Germany) w ith a total imprecision below 10%. Total TC concentration was determined by a sandwich ELISA with a total imprecision of 4 to 6% (intraassay imprecision ~3%) (133). After removal of the apo-TC with B12 coated beads, holo-TC was measured by the TC ELISA. The total im precision for measurement of holo-TC was ~8% (48), and the intra-assay imprecision was ~4% (1 34). Albumin and creatinine were measured on the Cobas Integra 800 (Roche Di agnostics, Indianapolis). Total imprecision was ~2 % for albumin and <3 % for creatinine. Homocysteine concentration was measur ed by the immunological method on the IMMUNLITE 2000 (Diagnostic Products Corporati on, California) (total imprecision <6%) (135) and MMA concentration was measured by slightly modified stable-isotope -dilution capillary gas chromatography mass-spectrometry (total imprecision <8%) (136).

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69 Statistical Methods Results are reported as mean SD with an alpha = 0.05 unless ot herwise noted. The overall p-value for time was obtained by the F-te st, which tests the nu ll hypothesis that the distribution of the dependent variable was the sa me at all time points. The Tukey method (137) of multiple comparisons was utilized for assessm ent of differences between time periods. A Least Significant Difference (LSD), as defi ned by the Tukey procedure, ensures that simultaneously, in every target population, paired di fference in means will be within +/LSD of the corresponding difference in sample means with 95% confidence. Results Mean baseline values for all analytes were within normal ranges, although some individuals had values outside the normal range (Table 1). Plasma albumin concentration fluctuated throughout the interven tion period suggesting a change in hydration status throughout day one and between the mornings of days 1, 2 and 3 (data not shown). Holo-transcobalamin, B12, and total-TC concentrations are reported as a ratio to albumin to adjust for diurnal changes in overall body protein concentration due to chan ges in hydration status. Unadjusted means for holo-TC, B12, and total-TC concen trations are reported in Table 2. All time-points are reported relative to baseline. Of all of the status i ndicator analytes, only hol o-TC and TC saturation changed significantly on day 1. Mean holo-TC concentration increased steadily after baseline and fluctuated throughout day 1. There were statistically significant increases in mean holo-TC concentr ation during the first 24 hours of the intervention; however, thes e small increases were not maintained. Mean holo-TC concentration reached a maximum value at hour 24, whic h was a significant increase relative to baseline and all other time points (F igure 2A). The mean percent increase from baseline also was greater at hour 24 than at all other all time point s with a 49% increase relative

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70 to baseline, and a 29% increase relative to hou r 12 (Figure 3). This peak at hour 24 was observed for almost all subjects, with an increas e of 22% or greater (22 to 85%) for all but one subject. By hour 48, mean holo-TC concentratio n decreased significantly relative to hour 24 (33%); however, it was still significantly greater than baseline (Figure 5-2A). Mean serum B12 concentration did not increas e significantly relative to baseline on day 1, although there were fluctuations in concentra tion throughout the day. At hour 24, mean serum B12 concentration was significantly greater than ba seline (Figure 5-2B). Overall, the percent change in B12 concentration was smaller than for holo-TC throughout the intervention period with ranges of -2 to 15% and -1 to 50%, respectively. Mean total-TC concentration did not change significantly during the study varying less than 6% from baseline at all time points (data not shown). Mean TC saturation began to increase significantly relative to baseli ne at hour 12.5, with the most significant increase at hour 24 (Figure 2C). As observed with holo-TC con centration, the mean TC saturation and percent change at hour 24 were signifi cantly greater than at all othe r time-points with 48% and 15% increases from baseline and hour 12.5, respectively (Figure 5-4). Among all subjects, the percent change from baseline ranged from 7 to 104% w ith 19 of 21 subjects having a value of 22% or greater. The ratio of holo-TC to B12 did not in crease significantly until hour 24 with absolute and percent increases of 0.15 and 32% respectively. The range for percent change in this ratio among all subjects was -7 to 109% with 15 of 21 subj ects having an increase of 23% or greater at hour 24. Discussion In this intervention study the changes in marker s of B12 status were measured on an hourly basis during and following administration three 9 g oral doses of B12. In previous studies the

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71 changes in response to similar B12 doses were measured after 24 hours; however, no data were collected prior to this time-point (28, 47, 104). Th e data from the present study indicate that a series of three 9 g doses of oral B12, given ove r 12 hours, led to small fluctuations in holo-TC concentration during the day 1 of the study followed by the previously observed maximum increase in holo-TC concentration 24 hours afte r the first B12 dose was given. There is a similarity in the overall pattern of change in holo-TC, B12 and TC saturation, with a gradual increase over the first day and the most pronounced increase occurring 24 hours after the initial B12 dose and 13 hours after the final B12 dose. The timing of B12 absorption and metabolism ma y explain the pattern of change observed in holo-TC concentration during th e first 12 hours of the interven tion. An increase in holo-TC concentration is first measurable in the blood af ter 3 to 4 hours after i ngestion and holo-TC can be taken up by cells within minutes (23). It is hypothesized that until ce lls are saturated with holo-TC, most of it is taken up so quickly that no major changes in blood levels would be observed initially. When intake is sufficient to sa turate the cells with B 12, significant changes in holo-TC can then be measured. The absolute and percentage increases in B12 concentration were smaller, occurred later, and were maintained longer than those for holo-TC This finding is not su rprising as total serum B12 consists primarily of holo-HC, and the sl ower rate of HC metabolism relative to TC metabolism leads to a slower overa ll turnover of serum B12 and a slower response to changes in intake (26, 138). When comparing these two me asures among the individual subjects, holo-TC had the most consistent pattern with only 1 subj ect not having a change of 20% or greater at hour 24. Additionally, the mean percen t change at hour 24 was three times that of B12. Holo-TC concentration is clearly a more sensitive indicato r of change in B12 inta ke and absorption than

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72 serum B12 concentration since it increases earl ier after supplementation, increased relatively more than serum B12 and decreased earlier post-supplementation ceased. Total-transcobalamin concentr ation did not change signifi cantly during the intervention period. Transcobalamin saturation increased in a similar manner to holo-TC (Figure 5-4). Both holo-TC concentration and TC saturation had comparable resu lts even when considering individual subjects. Of all subjects, 95% and 90% had increa ses of at least 22% at hour 24 for holo-TC and TC saturation, respectively. In a pr evious study, a larger ch ange in TC saturation (at hour 24) than for holo-TC was observed, which was due to a drop in total TC at this time point (47). No such conclusi on can be made from our data since no significant difference was observed. Since TC saturation is a calculated rather than a direct measure, the potential error in this value is greater than that for holo-TC concentration. Ther efore holo-TC concentration may be the better indicator of B12 absorption. This is the first study to mon itor hourly changes in holo-TC concentration in response to oral B12 intake. The most significant change in holo-TC concentra tion occurred at hour 24, indicating this is the optimal ti me post-dose at which to measure holo-TC. The three 9 g oral vitamin dose sequence used in this study was used to minimize passive absorption and maximize the amount of actively absorbed B12 (47, 104). This aspect of the protoc ol would be important in a clinical B12 absorption test, because it is the capacity to actively absorb B12 that is being assessed. Further studies evalua ting the necessity of three dose s and the exact timing of the doses are warranted. In conclusion, holo-TC increases measurably in response to administration of oral B12 within six hours with a maximum peak at 24 hour s. Our results indicate that a B12 absorption

79 CHAPTER 6 DISCUSSION Vitamin B12 is an essential water soluble vitamin functioning as a coenzyme for two metabolic processes, the conversion of methylmalonyl-CoA to succinyl-CoA as adenosylcobalamin and the remethylation of Hcy to methionine as methylcobalamin (13, 35). Absorption and utilization of B12 are dependent on adequate gastric HC l production to release food-bound B12, IF in the in testine for active transport of B12 into the enterocy te, and TC for uptake into body tissues. A defici ency of any of these compone nts can impair B12 metabolism and lead to deficiency even if diet ary intake is sufficient (139). The RDA for B12 is 2.4 g for adults (140). Older adults ( > 60 y) have an increased risk for B12 malabsorption due to an age related in creased risk for achlorhydria and auto-immune based pernicious anemia. The RDA fo r B12 in older adults is also 2.4 g/d, but it is recommended that synthetic B12 provided by su pplements or fortified foods be the primary source (140). Individuals with pernicious anemia are generally treated with IM B12 injections, although it has been reported that passive absorption of megadoses of oral B12 may be sufficient to meet dietary needs (141-143). Vitamin B12 is synthesized by microorganisms, pr esent in the intestinal microflora and is found naturally only in animal-derived foods. Cons equently, individuals who restrict their intake of some or all animal-derived foods limit thei r chances of consuming a diet that provides an adequate amount of vitamin B12. Consumption of B12-fortified foods or B12containing vitamin supplements can provide sufficient B12 fo r these individuals; however, it is estimated that ~ 60 % of the US population does not take supplements (144). Alth ough B12 is required in relatively small amounts, long term adherence to a B12-deficient diet can lead to a B12 deficiency and even moderate B12 deficiency can seriously impair health. Of greatest concern

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80 are individuals who consume diets with restricted intakes of animal-based foods and who do not take B12-containing vitamin supplements or cons ume B12 fortified foods. Studies comparing the B12 status of vegetarians and omnivores have led to the conclusion th at vegetarians are at greater risk for developing a B12 deficiency compared to omnivores (40, 81, 86, 145); however the majority of these studies have been conducted in Europe and therefore may not be applicable to the US population. Additionally they have included both supplement users and nonusers, making it difficult to interpret the effect of dietary B12 intake alone on status. It is estimated that B12 intake in the US exceeds the current RDA (2.4 g/d) leading to the conclusion that B12 dietary inadequacy is not a problem in the US (111, 146). The position of the American Dietetic Association is that approp riately planned vegetarian diets are healthful, nutritionally adequate, a nd provide health benefits in the prevention and treatment of certain diseases (110). The key to this statement is that a meat-free diet must be well planned to ensure that vitamin and mineral needs are met. The da ta from the current st udy, in addition to those from the Framingham Offspring study, and an investigation by Bor at al. suggest that consumption of the current RDA is insufficient to maintain normal B12 status in a significant percentage of young healthy adults (118, 119). A lthough these data do suggest that the current RDA is inadequate to maintain normal B12 status they are insufficient to provide a definitive estimation of a new RDA. In the current study, a FFQ was used to estimate B12 intake. While data generated from FFQs are adequate for obtaining information on relative frequency of consumption of nutrients, contribution of food cate gories to overall intake and estimating intake of key nutrients, FFQs do not generate data prec ise or specific enough to estimate a nutrient requirement. Future controlled metabolic studies designed to estimate the quantity of B12 intake

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81 at which B12 status is optimal are needed since controlled metabolic studies have proven to be highly useful in estimating other nutrient requirements (147, 148). Future studies assessing B12 status need to m easure multiple indicators of B12 status. One strength of the current series of studies was that numerous biom arkers were used to asses B12 status, rather than just one. Although the asse ssment of B12 status has traditionally been based on plasma or serum B12, approximately 5 to 10% of individuals with a plasma B12 concentration between 148 to 221 pmol/L, have been reported to have hematological or neurological abnormalities that responded to B12 supplementation (44, 97). Assessment of vitamin B12 status based on serum holo-TC concentr ation, a relatively new B12 status indicator, has been reported to be an earlier marker of changes in B12 status than total plasma B12 concentration. It has been suggested that meas urement of B12 and holo-TC concentrations in combination may be superior to either alone (27, 28, 81, 116, 149). Plasma homocysteine and serum MMA concentrations are functional indicators of B12 status and ar e inversely related to B12 concentration; however, only MMA concentr ation is specific for B12 status and is considered by some to be the most reliable B12 status indicator (35, 54). There is no clear consensus as to which particular B12 biomarker might be used as a gold standard; however, data from the current set of studi es suggest that a panel of B12 bi omarkers is preferable to any one status indicator for B12 status assessment. Additionally measurements at multiple time points over several days could help confirm a possible diagnosis of B12 deficiency, particularly in the case of holo-TC, which has been reported to be highly sensitive to ch anges in dietary B12 intake. The sensitivity of holo-TC has also led to the hypothesis that it could be used to assess B12 absorption (47, 104). In a previous study conduc ed by Bor et al. (104), it was reported that

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82 measurement of holo-TC 24 hours after ad ministration of a series of three 9 g doses of oral B12 identified individuals with B12 malabsorption. Individuals defined as B12 malabsorbers based on the Schilling test for B12 absorption had no si gnificant change in holoTC in contrast to a significant increase observed in normal controls. In the current set of studies, changes in holoTC and other markers of B12 status were m easured hourly with administration of three 9 g oral doses of B12, to determine whether any significant changes occur before 24 hours. A clear peak in holo-TC concentration was observed at hour 24 for all but one of the 22 subjects with only small fluctuations in holo-TC prior to that. Th is was the first study to monitor hourly changes in holo-TC in response to oral B12 suggesting a te st of B12 absorption ut ilizing holo-TC should involve measurement of holo-TC at baseline a nd 24 hours later. A limita tion of the current study was that only healthy individuals with normal B12 status were incl uded in this investigation. It is possible that saturatio n of cells might be necessary before an increase in holo-TC can be measured even in an individual with no B12 abso rption problems. Therefore, individuals with low B12 status may need more oral B12 and may have a later peak increase in holo-TC compared to individuals with normal B12 status. It is important to note that a B12 malabsorption test would only be run in an i ndividual with B12 deficiency; ther efore, future studies evaluating holo-TC as a measure of B12 absorption needs to compare the efficacy of changes in holo-TC as an index of B12 absorption in individuals with deficient versus normal B12 status. A final objective of this series of studies wa s to determine the effect of specific genenutrient interactions on B12 metabolism. Rare congen ital defects known to impair B12 metabolism and status include various mutations and post-translational changes that result in altering IF and TC protein structur e or a total lack of protein synthesis. Congenital errors in IF or TC lead to pernicious anemia; however e rrors evolving IF only im pair intestinal B12

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83 absorption and can be treated by lifelong IM admi nistration of B12, while errors involving TC lead to death early in life because B12 tran sport and uptake into body cells can not occur (150, 151). Perhaps less apparent than these severe ge netic defects, are polymorphisms that also may alter protein structure enough to impair function. One such polymorphism investigated in the present investigation was the TC 776G G polymorphism. In a previous study by our research group a significant effect of the polymorphism on holo-TC concentration was observed but no difference was detected in the current study. The small differences found between genotype groups in total-TC but not TC saturation suggest some small effect of the polymorphism however, there is likely to be no physiological impact of this pol ymorphism Previous studies focusing only on the effect of the polymorphism on a developing fetus have resulted in mixed findings, though continued investigations re lated to the potentia l association of B12-related polymorphisms and health-relate d consequences are warranted (128-130). In conclusion, based on data from this series of investigations it is clear that healthy individuals who do not take supplements may not be consuming adequate B12 to meet biological requirements, particularly those limiting some or all animal-based foods. Although moderate B12 deficiency may not result in overt symptoms, the associated increased risk for disease and birth defect-affected pregnancies provide an impetus for continued research focusing on determining the optimal B12 intake to maintain normal status. Early findings of the potential negative effect of the TC 776C G polymorphism on B12 metabol ism were not confirmed by the current data, and any future investigations should focus on the combined effects of multiple polymorphisms in genes involved in B12 metabolis m. Accurate detection and diagnosis of a B12 deficiency and its cause will help in th e prevention of related health problems including abnormal pregnancy outcomes. Although there is yet no consensus on a single gold standard

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84 test of B12 status, simultaneous measurement of two or more B12 biomarkers at several time points may be the best diagnostic approach. If existence of a B12 deficiency is established, further testing to determine if it is due to diet ary insufficiency or mala bsorption will aid in determining an appropriate treatment, includ ing changes in dietary B12 intake and/or supplementation. Data from the current investigati ons support the use of holo-TC as an indicator of B12 absorption though further re search is needed before a clin ically reliable test could be developed.

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85 APPENDIX A SUBJECT PHONE SCREENING FORM Introduction I am calling in regard to your interest in our nutrition study; do you ha ve a few minutes right now? This is a UF Nutrition department study and involves coming in one morning for about 1 hour for a fasting blood sample, we take about 1 oun ces of blood, and you only need to fast 8 hours. We will give you a breakfast snack right afterward, and then give a brief explanation of a food frequency questionnaire you will be taking home. You will be asked to mail it back in the provided envelope, and once we receive the ques tionnaire you would get pa id the $50. I just have to ask you some questions to see if you are eligible for our st udy and to get background information, OK? How old are you? Must be 18-49 Do you smoke? Must answer no Are you pregnant or breastfeeding ? Must answer no Do you take any prescription medications other than oral contraceptives ? Must answer no If not within the age range or if they answe r yes to any above questions end call with: I am very sorry, but you do not meet our exclus ion criteria, but thank you for your interest. Now I just have a few questions about your diet to see what specific category of our study you would fit in to. Please answer as best you can, estimates are ok and consider all instances of when you might eat the items I will ask about, even if only occasionally. Do you take a multi-vitamin, complex, red star nutritional yeast, or any other supplement or additive ever? If they take a multivitamin, B complex, red star nutritional yeast, complete the session through all diet info only. Conclude by confirming their name and saying This has been a preliminary screening call, your information will be reviewed by the principal investigator based on need, and our selection criteria at this time. If you are chosen you will be called again to schedule an appointment over the next two weeks. Thank you very much for your interest and your time. Do you eat breakfast cereals? ( If so) What Kind do you eat mostly? If they eat a 100% fortified cer eal or eats a 50% cereal daily complete the through all diet info but do not record. Conclude by confirming their name and saying This has been a preliminary screening call, your information will be reviewed by the principal investigator based on need, and our selection criteria at this time. If you are chosen you will be called again to schedule an appointment over the next two weeks. Thank you very much for your interest and your time. If the interviewee fulfills all se lection criteria continue with the questionnaire, record info on moderate/non-fortified cereal consumption

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86 Do you eat breakfast cereals? o Yes o No Name/Brand Quantity Frequency Are you a vegan, vegetarian or meat eater? Vegan this means you eat NO animal derived foods intentionally (if they eat small amt like in cake then OK) Vegetarian this means you eat NO bee f, chicken, turkey, pork, or fish How often do you eat Never Rarely (<1 x/mo) Occasionally (1-4 x/mo) Frequently (2-4 x/wk) Always (5-7 x/wk) Beef Chicken Turkey Pork Fish Eggs Cheese Cows Milk Yogurt Other Dairy Do you follow a restricted diet such as o No red meat o Lactose-free o Kosher o Weight loss o Weight gain o Low salt o Low fat o Low cholesterol o Low carbohydrate o Hypoallergenic (If so) How long have you consumed this type of diet? ________________________________________________________________________ Have you made any major dietary ch anges within the last 3 years? o No o Yes; How long ago did you make cha nges and what changes did you make? ________________________________________________________________________ NO YES Do you consume alcoholic beverages? How often/quantity Health Information

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87 I am going to ask you a few questions about your health to determine if you are eligible for our study. I will be recording this information, but it will be kept confidential and is this ok with you? _______ Height: Weight: Have you do you currently have any of the following? NO YES Alcoholism Anemia Blood clots Bronchitis Cystic Fibrosis Dermatitis Diabetes Eating disorders/Chronic nausea or vomiting Food allergy Gall bladder disease GI problems/ Lactose intolerance Gout Migraines Hemorrhoids Hepatitis/Liver disease Heart disease/High cholesterol/High blood pressure HIV Kidney disease Neurological disorder Obesity Seizures/Stroke Thyroid problem Tumors/Cancer Ulcers Other Have you been hospitalized within the last 5 years? Cause Do you have a history of more than 1 miscarriage? o Yes o No If you are selected to participate in this study are you willing to sign an informed consent understanding we have access to medical information on you? o Yes o No Demographic Information What is your birth date? _______/_______/_________

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88 Month Day Year How would you describe your race or ethnic background? o White o Black or African American o American Indian or Alaska Native o Hispanic or Latino o Asian o Native Hawaiian or Other Pacific Islander o Other _________________________________________________ What is the highest level of school or training that you have completed? [Circle only one response] Grade school 01 02 03 04 05 06 07 08 High school 09 10 11 12 Technical school or college 13 14 15 16 Graduate or professional 17 18 19 20+ Dont know X Marital status? o Single/never married o Married o Separated o Divorced o Widowed Are you a full-time or part-time student? Are you employed? o Full time o Yes o Part time o No o Not a student o Student employee

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89 Contact Information Name of person and phone number to call in case of an emergency if you are invited to participate in this study: ______________________________________________________________________________ If we need to contact you, and can not reach you where/with who can a message be left? ______________________________________________________________________________ How did you hear about our study? _______________________________________________ Name M / F Last First Middle Street Apt. # Address City Zip code Phone Day Evening Cell E-mail

103 BIOGRAPHICAL SKETCH Kristina M von Castel Robert s was born in Camden, New Jersey in 1975. She lived in Glenside and Penllyn, Pennsylvania from 1981 until she graduated from Springside School for girls in 1993. She graduated from the University of Florida in 2000 with a bachelor of science degree in animal science, with a specialization in animal bi ology. She was employed by the University of Florida Racing Laboratory until she entered her Ph.D. program in nutritional sciences in the fall of 2002 under the Davis Alum ni fellowship. During her doctoral program she studied under the tutelage of Dr. Lynn B. Bailey in the field of folate and vitamin B12 nutrition and metabolism. Upon graduation she w ill continue her care er in academia.